Food ingredients produced from high amylose wheat

ABSTRACT

Provided are food and drink ingredients produced from wheat grain which has a low level (2-30%) of total starch branching enzyme II activity and an amylose content in the starch of at least 50% (w/w), and processes for producing and using the ingredients. Also provided are foods produced from the ingredients which may be used in humans to improve one or more parameters of metabolic health, bowel health or cardiovascular health.

This application is a continuation of U.S. application Ser. No.15/440,652, filed Feb. 23, 2017, which is a continuation of U.S.application Ser. No. 13/883,456, filed Aug. 6, 2013, now U.S. Pat. No.9,585,413, issued Mar. 7, 2017, which is a § 371 national stage of PCTInternational Application No. PCT/AU2011/001426, filed Nov. 4, 2011,claiming the benefit of U.S. Provisional Application No. 61/410,288,filed Nov. 4, 2010, the contents of each of which are herebyincorporated by reference in their entirety.

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“200305_82306-AAA-PCT-US_Substitute_Sequence_Listing_CAS.txt,” which is109 kilobytes in size, and which was created Mar. 5, 2020 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Mar. 5, 2020 aspart of this application.

FIELD

The specification describes methods of obtaining hexaploid wheat plantshaving high amylose starch and the use of such plants, and particularlygrain or starch therefrom in a range of food and non-food products.

BACKGROUND

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

In the last decade, much has been learnt about the molecular, geneticand cellular events underpinning plant life cycles and plant production.One particularly important plant product is wheat grain. Wheat grain isa staple food in many countries and it supplies at least 20% of the foodkilojoules for the total world population. Starch is the major componentof wheat grain and is used in a vast range of food and non-foodproducts. Starch characteristics vary and they play a key role indetermining the suitability of wheat starch for a particular end use.Despite this huge global consumption and despite an increased awarenessof the importance of starch functionality on end product quality,research on genetic variation in wheat and its precise impact on starchcharacteristics lags behind that for other commercially important plantcrops.

Bread wheat (Triticum aestivum) is a hexaploid having three pairs ofhomoeologous chromosomes defining genomes A, B and D. The endosperm ofgrain comprises 2 haploid complements from a maternal cell and 1 from apaternal cell. The embryo of wheat grain comprises one haploidcomplement from each of the maternal and paternal cells. Hexaploidy hasbeen considered a significant obstacle in researching and developinguseful variants of wheat. In fact, very little is known regarding howhomoeologous genes of wheat interact, how their expression is regulated,and how the different proteins produced by homoeologous genes workseparately or in concert.

Cereal starch is made up of two glucose polymers, amylose andamylopectin. The ratio of amylose to amylopectin appears to be a majordeterminant in (i) the health benefit of wheat grain and wheat starchand (ii) the end quality of products comprising wheat starch.

Amylose is an essentially linear polymer of α-1,4 linked glucose units,while amylopectin is highly branched with α-1,6 glucosidic unit bondslinking linear chains.

High amylose starches are of particular interest for their healthbenefits. Foods comprising high amylose have been found inter alia to benaturally higher in resistant starch, a form of dietary fibre. RS isstarch or starch digestive products that are not digested or absorbed inthe small intestine. Resistant starch is increasingly seen to have animportant role in promoting intestinal health and in protecting againstdiseases such as colorectal cancer, type II diabetes, obesity, heartdisease and osteoporosis. High amylose starches have been developed incertain grains such as maize and barley for use in foods as a means ofpromoting bowel health. The beneficial effects of resistant starchresult from the provision of a nutrient to the large bowel wherein theintestinal microflora are given an energy source which is fermented toform inter alia short chain fatty acids. These short chain fatty acidsprovide nutrients for the colonocytes, enhance the uptake of certainnutrients across the large bowel and promote physiological activity ofthe colon. Generally, if resistant starches or other dietary fibre arenot provided to the colon it becomes metabolically relatively inactive.Thus high amylose products have the potential to facilitate increasedconsumption of fibre. Some of the potential health benefits of consuminghigh amylose wheat grains or their products such as starch include itsrole in regulating sugar and insulin and lipid levels, promotingintestinal heath, producing food of lower calorie value that promotesatiety, improving laxation, water volume of faeces, promoting growth ofprobiotic bacteria, and enhancing faecal bile acid excretion.

Most processed starchy foods contain very little RS. The breads madeusing wild-type wheat flour and a conventional formulation and bakingprocess contained <1% RS. In comparison, breads baked using the sameprocess and storage conditions but containing the modified high amylosewheats had levels of RS as much as 10-fold higher (see InternationalPublication No. WO 2006/069422). Legumes, which are one of the few richsources of RS in the human diet, contain levels of RS that are normally<5%. Therefore, consumption of the high amylose wheat bread in amountsnormally consumed by adults (e.g. 200 g/d) would readily supply at least5-12 g of RS. Thus, incorporation of the high amylose wheat into foodproducts has the potential to make a considerable contribution todietary RS intakes of developed nations, where average daily intakes ofRS are estimated to be only about 5 g.

Starch is widely used in the food, paper and chemical industries. Thephysical structure of starch can have an important impact on thenutritional and handling properties of starch for food or non-food orindustrial products. Certain characteristics can be taken as anindication of starch structure including the distribution of amylopectinchain length, the degree and type of crystallinity, and properties suchas gelatinisation temperature, viscosity and swelling volume. Changes inamylopectin chain length may be an indicator of altered crystallinity,gelatinisation or retrogradation of the amylopectin.

Whilst chemically or otherwise modified starches can be used in foodsthat provide functionality not normally afforded by unmodified sources,such processing has a tendency to either alter other components of valueor carry the perception of being undesirable due to processes involvedin modification. Therefore it is preferable to provide sources ofconstituents that can be used in unmodified form in foods.

Starch is initially synthesized in plants in chloroplasts ofphotosynthesizing tissues such as leaves, in the form of transitorystarch. This is mobilized during subsequent dark periods to supplycarbon for export to sink organs and energy metabolism, or for storagein organs such as seeds or tubers. Synthesis and long-term storage ofstarch occurs in the amyloplasts of the storage organs, such as theendosperm, where the starch is deposited as semicrystalline granules upto 100 μm in diameter. Granules contain both amylose and amylopectin,the former typically as amorphous material in the native starch granulewhile the latter is semicrystalline through stacking of the linearglucosidic chains. Granules also contain some of the proteins involvedin starch biosynthesis.

The synthases of starch in the endosperm is carried out in fouressential steps. ADP-glucose pyrophosphorylase (ADGP) catalyses thesynthesis of ADP-glucose from glucose-1-phosphate and ATP. Starchsynthases then promote the transfer of ADP-glucose to the end of anα-1,4 linked glucose unit. Thirdly, starch branching enzymes (SBE) formnew α-1,6 linkages in α-polyglucans. Starch debranching enzymes (SDBE)then remove some the branch linkages through a mechanism that has notbeen fully resolved.

While it is clear that at least these four activities are required fornormal starch granule synthesis in higher plants, multiple isoforms ofenzymes taking part in one of the four activities are found in theendosperm of higher plants. Specific roles for some isozymes have beenproposed on the basis of mutational analysis or through the modificationof gene expression levels using transgenic approaches (Abel et al.,1996; Jobling et al., 1999; Schwall et al., 2000). However, the precisecontributions of each isoform of each activity to starch biosynthesisare still not known, and these contributions appear to differ markedlybetween species.

In the cereal endosperm, two isoforms of ADP-glucose pyrophosphorylase(ADGP) are present, one form within the amyloplast, and one form in thecytoplasm. Each form is composed of two subunit types. The shrunken(sh2) and brittle (bt2) mutants in maize represent lesions in large andsmall subunits respectively.

Some efforts have focused on starch synthase enzymes to investigatestrategies to modulate the amylose/amylopectin ratio in wheat (seeSestili et al. 2010).

Four classes of starch synthase (SS) are found in the cereal endosperm,an isoform exclusively localised within the starch granule(granule-bound starch synthase (GBSS)) two forms that are partitionedbetween the granule and the soluble fraction (SSI and SSII) and a fourthform that is entirely located in the soluble fraction (SSIII). GBSS hasbeen shown to be essential for amylose synthesis and mutations in SSIIand SSIII have been shown to alter amylopectin structure.

A mutant wheat plant entirely lacking the SGP-1 (SSIIa) protein wasproduced by crossing lines which were lacking the A, B and D genomespecific forms of SGP-1 (SSII) protein (Yamamori et al., 2000).Examination of the SSII null seeds showed that the mutation resulted inalterations in amylopectin structure, deformed starch granules, and anelevated relative amylose content to about 30-37% of the starch, whichwas an increase of about 8% over the wild-type level (Yamamori et al.,2000). Amylose was measured by colorimetric measurement, amperometrictitration (both for iodine binding) and a concanavalin A method. Starchfrom the SSII null mutant exhibited a decreased gelatinisationtemperature compared to starch from an equivalent, non-mutant plant.Starch content was reduced from 60% in the wild-type to below 50% in theSSII-null grain.

In maize, the dull1 mutation causes decreased starch content andincreased amylose levels in endosperm, with the extent of the changedepended on the genetic background, and increased degree of branching inthe remaining amylopectin. The gene corresponding to the mutation wasidentified and isolated by a transposon-tagging strategy using thetransposon mutator (Mu) and shown to encode the enzyme designated starchsynthase II (SSII). The enzyme is now recognized as a member of theSSIII family in cereals. Mutant endosperm had reduced levels of SBEIIaactivity associated with the dull1 mutation. It is not known if thesefindings are relevant to other cereals.

Lines of barley having an elevated proportion of amylose in grain starchhave been identified. These include High Amylose Glacier (AC38) whichhas a relative amylose content of about 45%, and chemically inducedmutations in the SSIIa gene of barley which raised levels of amylose inkernel starch to about 65-70% (WO 02/37955 A1; Morell et al., 2003). Thestarch showed reduced gelatinisation temperatures.

Two main classes of SBEs are known in plants, SBEI and SBEII. SBEII canbe further categorized into two types in cereals, SBEIIa and SBEIIb.Additional forms of SBEs are also reported in some cereals, a putative149 kDa SBEI from wheat and a 50/51 kDa SBE from barley.

Sequence alignment reveals a high degree of sequence similarity at boththe nucleotide and amino acid levels and allows the grouping into theSBEI, SBEIIa and SBEIIb classes. SBEIIa and SBEIIb generally exhibitaround 80% nucleotide sequence identity to each other, particularly inthe central regions of the genes.

In maize and rice, high amylose phenotypes have been shown to resultfrom lesions in the SBEIIb gene, also known as the amylose extender (ae)gene (Boyer and Preiss, 1981, Mizuno et al., 1993; Nishi et al., 2001).In these SBEIIb mutants, endosperm starch grains showed an abnormalmorphology, amylose content was significantly elevated, the branchfrequency of the residual amylopectin was reduced and the proportion ofshort chains (<DP17, especially DP8-12) was lower. Moreover, thegelatinisation temperature of the starch was increased. In addition,there was a significant pool of material that was defined as“intermediate” between amylose and amylopectin (Boyer et al., 1980,Takeda et al 1993b). In contrast, maize plants mutant in the SBEIIa genedue to a mutator (Mu) insertional element and consequently lackingSBEIIa protein expression were indistinguishable from wild-type plantsin the branching of endosperm starch (Blauth et al., 2001), althoughthey were altered in leaf starch. In both maize and rice, the SBEIIa andSBEIIb genes are not linked in the genome.

SBEIIa, SBEIIb and SBEI may also be distinguished by their expressionpatterns, both temporal and spatial, in endosperm and in other tissues.SBEI is expressed from mid-endosperm development onwards in wheat andmaize (Morell et al., 1997). In contrast, SBEIIa and SBEIIb areexpressed from an early stage of endosperm development. In maize, SBEIIbis the predominant form in the endosperm whereas SBEIIa is present athigh expression levels in the leaf (Gao et al., 1997). In rice, SBEIIaand SBEIIb are found in the endosperm in approximately equal amounts.However, there are differences in timing and tissues of expression.SBEIIa is expressed at an earlier stage of seed development, beingdetected at 3 days after flowering, and was expressed in leaves, whileSBEIIb was not detectable at 3 days after flowering and was mostabundant in developing seeds at 7-10 days after flowering and was notexpressed in leaves. In wheat endosperm, SBEI (Morell et al, 1997) isfound exclusively in the soluble fraction, while SBEIIa and SBEIIb arefound in both soluble and starch-granule associated fractions (Rahman etal., 1995).

Very high amylose varieties of maize have been known for some time. Lowamylopectin starch maize which contains very high amylose content (>90%)was achieved by a considerable reduction in the SBEI activity togetherwith an almost complete inactivation of SBEII activity (Sidebottom etal., 1998).

In potato, down regulation of the main SBE in tubers (SBE B, equivalentto SBEI) by antisense methods resulted in some novel starchcharacteristics but did not alter the amylose content (Safford et al.,1998). Antisense inhibition of the less abundant form of SBE (SBE A,analogous to SBEII in cereals) resulted in a moderate increase inamylose content to 38% (Jobling et al., 1999). However, the downregulation of both SBEII and SBEI gave much greater increases in therelative amylose content, to 60-89%, than the down-regulation of SBEIIalone (Schwall et al., 2000).

International Publication No. WO 2005/001098 and InternationalPublication No. WO 2006/069422 describe inter alia transgenic hexaploidwheat comprising exogenous duplex RNA constructs that reduce expressionof SBEIIa and/or SBEIIb in the endosperm. Grain from transgenic linescarried either no SBEIIa and/or SBEIIb protein or reduced proteinlevels. A loss of SBEIIa protein from endosperm was associated withincreased relative amylose levels of more than 50%. A loss of SBEIIbprotein levels did not appear to substantially alter the proportion ofamylose in grain starch. It was proposed but not established that aSBEIIa and/or SBEIIb triple null mutant substantially lacking expressionof SBEIIa and SBEIIb proteins would result in further elevations ofamylose levels. However, it was not known or predictable from the priorart how many mutant alleles of SBEIIa and/or SBEIIb would be required toprovide high amylose levels of at least 50% as a proportion of the totalstarch. It was also unknown whether the grain of triple null genotypeswould be viable or whether the wheat plants would be fertile.

There is a need in the art for improved high amylose wheat plants andfor methods of producing same.

SUMMARY

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

As used herein the singular forms “a”, “an” and “the” include pluralaspects unless the context clearly dictates otherwise. Thus, forexample, reference to “a mutation” includes a single mutation, as wellas two or more mutations; reference to “a plant” includes one plant, aswell as two or more plants; and so forth.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Genes and other genetic material (e.g. mRNA, constructs etc) arerepresented in italics and their proteinaceous expression products arerepresented in non-italicised form. Thus, for example, SBEIIa is anexpression product of SBEIIa.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A sequence listing is provided after the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described.

The present invention provides a range of wheat plants having modifiedstarch characteristics.

In one embodiment, the invention provides wheat grain (Triticumaestivum) comprising an endosperm and a low level or activity of totalSBEII protein or SBEIIa protein that is 2% to 30% of the level oractivity of total SBEII or SBEIIa protein in a wild-type wheat grain,and wherein the grain comprises an amylose content of at least 50%(w/w), or at least 60% (w/w), or at least 67% (w/w) as a proportion ofthe total starch in the grain.

In one embodiment, the invention provides wheat grain comprising anembryo and starch, wherein the embryo comprises two identical alleles ofan SBEIIa-A gene, two identical alleles of an SBEIIa-B gene and twoidentical alleles of an SBEIIa-D gene, wherein each of the SBEIIa genesgives rise to an amount of protein (w/w) or a protein having SBEIIaactivity which is lower than the corresponding wild-type gene, and atleast one of said genes comprises a point mutation, wherein the starchcomprises amylose such that the grain has an amylose content of at least50% (w/w) as a proportion of the extractable starch of the grain.

In one embodiment, the invention provides wheat grain comprising anembryo, starch and one, two or three SBEIIa proteins, said embryocomprising two identical alleles of an SBEIIa-A gene, two identicalalleles of an SBEIIa-B gene and two identical alleles of an SBEIIa-Dgene, wherein the starch has an amylose content of at least 50% (w/w) asa proportion of the extractable starch of the grain, and wherein atleast one of the SBEIIa proteins is produced in the developing wheatendosperm and has starch branching enzyme activity.

In some embodiments, the amount and activity of the SBEIIa protein arereduced. Thus, for example, a grain of the invention may comprise areduced amount of SBEIIa protein (w/w) which has reduced SBEIIaactivity.

In various embodiments, the level or activity of total SBEII or SBEIIaprotein in the grain is less than 2% or 2% to 15%, or 3% to 10%, or 2%to 20% or 2% to 25% of the level or activity of total SBEII or SBEIIaprotein in the wild-type grain.

In some embodiments, the amount or activity of the SBEIIa protein in thegrain is less than 2% of the amount or activity of SBEIIa protein in awild-type wheat grain.

In another aspect, the grain is from hexaploid wheat.

In one embodiment, the grain is from hexaploid wheat and comprises anembryo, wherein the embryo comprises a loss of function mutation in eachof 5 to 12 alleles of endogenous SBEII genes selected from the groupconsisting of SBEIIa-A, SBEIIa-B, SBEIIa-D, SBEIIb-A, SBEIIb-B andSBEIIb-D. In one particular, said 5 to 12 alleles including 4, 5 or 6SBEIIa alleles each comprise a loss of function mutation. In anotherparticular, when the number of SBEIIa alleles comprising a loss offunction mutation is only 4 then the number of SBEIIb alleles comprisinga loss of function mutation is 6. In another embodiment, when the numberof SBEIIa alleles comprising a loss of function mutation is 6 then atleast two SBEIIb alleles comprise a partial loss of function mutation.In a further embodiment, the hexaploid wheat embryo has no null allelesof SBEIIb genes, or only 1, only 2, only 3, only 4, only 5 or 6 nullalleles of SBEIIb genes.

In a further embodiment, the hexaploid wheat embryo has only 2, only 3,only 4 or only 5 null alleles of SBEIIa genes.

In some embodiments, the hexaploid wheat embryo has 6 null alleles ofSBEIIa genes.

In some embodiments, the grain or embryo has only 1 null SBEIIa gene.

In some embodiments, the grain or embryo has only 2 null SBEIIa genes.

In a further embodiment, the hexaploid wheat embryo has no null allelesof SBEIIb genes, or only 1, only 2, only 3, only 4, only 5 or 6 nullalleles of SBEIIb genes.

In yet another embodiment, the null alleles of the SBEIIa or SBEIIbgenes are on the A genome, B genome, D genome, A and B genomes, A and Dgenomes, A and D genomes, or all three of the A, B and D genomes.

In yet another embodiment, the hexaploid wheat embryo comprises 0, 1, 2,3, 4, 5, or 6 partial loss of function alleles of SBEIIa genes. In somecases the partial loss of function allele of the SBEIIa gene is on the Agenome, B genome, D genome, A and B genomes, A and D genomes, A and Dgenomes, or all three of the A, B and D genomes.

Additionally, in some embodiments, the hexaploid wheat embryo comprises0, 1, 2, 3, 4, 5, or 6 partial loss of function alleles of SBEIIb genes.In some cases the partial loss of function allele of the SBEIIb gene ison the A genome, B genome, D genome, A and B genomes, A and D genomes, Aand D genomes, or all three of the A, B and D genomes.

In other embodiments, the partial loss of function alleles of the SBEIIaor SBEIIb genes are on the A genome, B genome, D genome, A and Bgenomes, A and D genomes, A and D genomes, or all three of the A, B andD genomes.

In another embodiment, the hexapoloid wheat embryo comprises 5 SBEIIaalleles each comprising a null or partial loss of function mutation and1 SBEIIa allele which is wild-type.

In another embodiment, the grain is from tetraploid wheat.

In another embodiment, the present invention provides wheat grain fromtetrapoloid wheat wherein the grain comprises an endosperm and a lowlevel or activity of total SBEII protein or SBEIIa protein that is 2% to30% of the level or activity of total SBEII or SBEIIa protein in awild-type wheat grain, and wherein the grain comprises an amylosecontent of at least 50% (w/w), or at least 60% (w/w), or at least 67%(w/w) as a proportion of the total starch in the grain.

In some embodiments, wherein the embryo comprises a loss of functionmutation in each of 5 to 8 alleles of endogenous SBEII genes selectedfrom the group consisting of SBEIIa-A, SBEIIa-B, SBEIIb-A and SBEIIb-B,said 5 to 8 alleles including 2, 3 or 4 SBEIIa alleles each comprising aloss of function mutation, and wherein when the number of SBEIIa allelescomprising a loss of function mutation is only 2 then the number ofSBEIIb alleles comprising a loss of function mutation is 4, and when thenumber of SBEIIa alleles comprising a loss of function mutation is 4then at least one, preferably at least two such alleles comprise apartial loss of function mutation.

In some embodiments, the embryo has only 2, or only 3, null alleles ofSBEIIa genes.

In one particular embodiment, the tetraploid wheat embryo has no nullalleles of SBEIIb genes, or only 1, only 2, only 3, or 4, null allelesof SBEIIb genes.

In some embodiments, the one SBEIIb protein is encoded by the A genome,the B, genome or D genome, or the two SBEIIb proteins are encoded by theA and B genomes, A and D, genomes, or B and D genomes.

In some embodiments, the null mutation is independently selected fromthe group consisting of a deletion mutation, an insertion mutation, asplice-site mutation, a premature translation termination mutation, anda frameshift mutation.

In some embodiments, the null alleles of the SBEIIa or SBEIIb genes areon the A genome, B genome, or both of the A and B genomes.

In other embodiments, the tetraploid wheat embryo comprises 0, 1, 2, 3or 4, 5, or 6 partial loss of function alleles of SBEIIa genes.

In other embodiments, the embryo comprises 0, 1, 2, 3 or 4 partial lossof function alleles of SBEIIb genes.

In yet another embodiment, the partial loss of function alleles of theSBEIIa or SBEIIb genes are on the A genome, B genome, or both of the Aand B genomes.

In some embodiments, the embryo is homozygous for mutant alleles in eachof 2 or 3 SBEIIa genes and/or each of 2 or 3 SBEIIb genes.

In other embodiments, the embryo is heterozygous for each of 2 or 3SBEIIa genes and/or each of 2 or 3 SBEIIb genes.

Usefully, in various embodiments of the present invention the graincomprises both null alleles and partial loss of function alleles ofSBEIIa and/or SBEIIb, wherein each of the null alleles is located on adifferent genome than each of the partial loss of function alleles.

In some embodiments relating to the null alleles, each null mutation isindependently selected from the group consisting of a deletion mutation,an insertion mutation, a splice-site mutation, a premature translationtermination mutation, and a frameshift mutation. In an embodiment, oneor more of the null mutations are non-conservative amino acidsubstitution mutations or a null mutation has a combination of two ormore non-conservative amino acid substitutions. In this context,non-conservative amino acid substitutions are as defined herein. Thegrain may comprise mutations in each of two SBEIIa genes, each of whichare null mutations, and an amino acid substitution mutation in a thirdSBEIIa gene, wherein each of the null mutations are preferably prematuretranslation termination mutations or deletion mutations, or onepremature translation termination mutation and one deletion mutation,and the amino acid substitution mutation is either a conservative aminoacid substitution or preferably a non-conservative amino acidsubstitution.

In some broad embodiments, the grain of the present invention includesone or more null mutations or partial loss of function mutations whichare amino acid substitution mutations, which are independentlynon-conservative or conservative amino acid substitutions.

In some embodiments, the grain of the present invention comprises onepoint mutation, which is an amino acid substitution mutation.

In some embodiments of the invention, one of the SBEIIa A, SBEIIa-B orSBEIIa-D genes comprises a point mutation such that the protein encodedby said gene lacks starch branching enzyme activity.

In some embodiments, the grain of the present invention has null alleleswhich are deletion mutations in the B and D genomes which delete atleast part of the SBEIIa-B and SBEIIa-D genes, respectively and whereinthe SBEIIa-A gene comprises the point mutation; or having null alleleswhich are deletion mutations in the A and D genomes which delete atleast part of the SBEIIa-A and SBEIIa-D genes, respectively and whereinthe SBEIIa-B gene comprises the point mutation; or having null alleleswhich are deletion mutations in the A and B genomes which delete atleast part of the SBEIIa-A and SBEIIa-B genes, respectively and whereinthe SBEIIa-D gene comprises the point mutation.

In some embodiments of the invention, the embryo comprises 6 SBEIIballeles of which at least one has a loss of function mutation.

In some embodiments of the invention, the embryo has no null alleles ofSBEIIb genes, or only 2, only 4 or 6 null alleles of SBEIIb genes.

In some embodiments, the grain comprises a null mutation which is adeletion mutation in the A, B or D genome, which deletes at least partof an SBEIIa gene and at least a part of an SBEIIb gene, preferablywhich deletes the whole of the SBEIIa gene, and/or the SBEIIb gene.

In some embodiments, the grain of the invention comprises a nullmutation which is a deletion mutation in the B genome which deletes atleast part of the SBEIIa-B gene and at least a part of the SBEIIb-Bgene, preferably which deletes the whole of the SBEIIa-B gene and/or theSBEIIb-B gene; or comprising a null mutation which is a deletionmutation in the D genome which deletes at least part of the SBEIIa-Dgene and at least a part of an SBEIIb-D gene, preferably which deletesthe whole of the SBEIIa-D gene and/or the SBEIIb-D gene; or comprising anull mutation which is a deletion mutation in the B genome which deletesat least part of the SBEIIa-A gene and at least a part of the SBEIIb-Agene, preferably which deletes the whole of the SBEIIa-A gene and/or theSBEIIb-A gene.

In illustrative examples, grain is provided wherein the allelescomprising a partial loss of function mutation each express an SBEIIa orSBEIIb enzyme which in amount and/or activity corresponds to 2% to 60%,or 10% to 50%, of the amount or activity of the corresponding wild-typeallele.

In some embodiments, the grain comprises at least one SBEIIa proteinwhich has starch branching activity when expressed in developingendosperm, the protein being present in an amount or having starchbranching enzyme activity of between 2% to 60%, or between 10% to 50%,or between 2% to 30%, or between 2% to 15%, or between 3% to 10%, orbetween 2% to 20% or between 2% to 25% of the amount or activity of thecorresponding protein in a wild-type wheat grain.

In some embodiments of the invention, the amount or activity of totalSBEII protein in the grain is less than 60%, preferably less than 2%, ofthe amount or activity of total SBEII protein in a wild-type wheatgrain.

In some embodiments of the invention, there is no SBEIIa proteinactivity in the grain.

Specifically, in some embodiments, the grain is non-transgenic i.e. doesnot comprise any transgene, or in a more specific embodiment does notcomprise an exogenous nucleic acid that encodes an RNA which reducesexpression of an SBEIIa gene i.e if it comprises a transgene, thattransgene encodes an RNA other than an RNA which reduces expression ofan SBEIIa gene. Such RNAs include RNAs which encode proteins that conferherbicide tolerance, disease tolerance, increase nutrient usageefficiency, or drought or other stress tolerance, for example.

In some embodiments, the grain has only one SBEIIa protein as determinedby Western blot analysis, and wherein the protein is encoded by one ofthe SBEIIa A, SBEIIa-B and SBEIIa-D genes and has reduced starchbranching enzyme activity when produced in developing endosperm whencompared to an SBEIIa protein encoded by the corresponding wild-typegene.

In some embodiments, the SBEIIa protein has an altered mobility relativeto its corresponding wild-type SBEIIa protein, as determined by affinitygel electrophoresis on gels containing starch.

In some embodiments, the grain lacks detectable SBEIIa protein asdetermined by Western blot analysis.

In some embodiments, the embryo comprises only one or only two SBEIIbproteins which have starch branching enzyme activity when produced indeveloping endosperm, or only one or only two SBEIIb proteins which aredetectable by Western blot analysis.

In relation to loss of function mutations, in some embodiments, at leastone, more than one, or all of the loss of function mutations are i)introduced mutations, ii) were induced in a parental wheat plant or seedby mutagenesis with a mutagenic agent such as a chemical agent,biological agent or irradiation, or iii) were introduced in order tomodify the plant genome.

In another illustrative embodiment, the grain comprises an exogenousnucleic acid which encodes an RNA which reduces expression of an SBEIIagene, an SBEIIb gene, or both.

As determined herein, grain is provided in some particular embodimentswherein the grain has a germination rate of about 70% to about 90%, orabout 90% to about 100% relative to the germination rate of a control orwild type grain under standard conditions. The standard conditions arepreferably as defined herein.

In one particular embodiment, the SBEII activity or SBEIIa activity isdetermined by assaying the enzymatic activity in grain while it isdeveloping in a wheat plant, or by assaying the amount of SBEII proteinsuch as SBEIIa protein in harvested grain by immunological or othermeans.

In another aspect, the present invention provides grain, wherein thestarch of the grain is at least 50% (w/w), or at least 60% (w/w), or atleast 67% (w/w) amylose as a proportion of the total starch and ischaracterised by one or more of:

-   -   (i) comprising 2% to 30% of the amount of SBEII or SBEIIa        relative to wild-type wheat starch granules or starch;    -   (ii) comprising at least 2% resistant starch;    -   (iii) comprising a low relative glycaemic index (GI);    -   (iv) comprising low relative amylopectin levels;    -   (v) distorted starch granules;    -   (vi) reduced granule birefringence;    -   (vii) reduced swelling volume;    -   (viii) modified chain length distribution and/or branching        frequency;    -   (ix) delayed end of gelatinisation temperature and higher peak        temperature;    -   (x) reduced viscosity (peak viscosity, pasting temperature,        etc.);    -   (xi) increased molecular weight of amylopectin; and/or    -   (xii) modified % crystallinity % A-type or B-type starch,        relative to a wild-type wheat starch granules or starch.

In some embodiments, the grain is comprised in a wheat plant.

In other embodiments, the grain is developing grain, or mature,harvested grain. Preferably the quantity of grain is at least 1 kgweight, or at least 1 tonne weight.

Conveniently, the grain is processed so that it is no longer capable ofgerminating, such as kibbled, cracked, par-boiled, rolled, pearled,milled or ground grain.

In another aspect the present invention provides a wheat plant which iscapable of producing the grain as defined herein including graincomprising an endosperm and a low level or activity of total SBEIIprotein or SBEIIa protein that is 2% to 30% of the level or activity oftotal SBEII or SBEIIa protein in a wild-type wheat grain, and whereinthe grain comprises an amylose content of at least 50% (w/w), or atleast 60% (w/w), or at least 67% (w/w) as a proportion of the totalstarch in the grain.

In one particular, the wheat plant is both male and female fertile.

In one embodiment, the wheat plant is bread wheat such as Triticumaestivum L. ssp. aestivum or durum wheat. In other embodiments, thewheat plant is characterised by one or more features of the grain asdescribed herein, preferably including the numbers and types of SBEIIaand SBEIIb mutations as described herein. All combinations of suchfeatures are provided.

In another embodiment, the invention provides wholemeal or flour oranother food ingredient such as purified starch produced from the grainas defined herein including grain comprising an endosperm and a lowlevel or activity of total SBEII protein or SBEIIa protein that is 2% to30% of the level or activity of total SBEII or SBEIIa protein in awild-type wheat grain, and wherein the grain comprises an amylosecontent of at least 50% (w/w), or at least 60% (w/w), or at least 67%(w/w) as a proportion of the total starch in the grain. The wholemeal,flour or other food ingredient may be refined by fractionation,bleaching, heat treatment to stabilise the ingredient, treated withenzymes or blended with other food ingredients such as wholemeal orflour from a wild-type wheat. The flour is preferably white flour,having specifications as known in the art of baking. In a preferredembodiment, the wholemeal, flour or other food is packaged ready forsale as a food ingredient, which package may include instructions ofrecipes for its use.

The present invention further contemplates wheat starch granules orwheat starch produced from the subject grain. In some embodiments, thestarch granules or wheat starch comprise at least 50% (w/w), or at least60% (w/w), or at least 67% (w/w) amylose as a proportion of the starch,and are further characterised by one of more of the features:

-   -   (i) comprising 2% to 30% of the amount of SBEII or SBEIIa        relative to wild-type wheat starch granules or starch;    -   (ii) comprising at least 2% resistant starch;    -   (iii) comprising a low relative glycaemic index (GI);    -   (iv) comprising low relative amylopectin levels;    -   (v) distorted starch granules;    -   (vi) reduced granule birefringence;    -   (vii) reduced swelling volume;    -   (viii) modified chain length distribution and/or branching        frequency;    -   (ix) delayed end of gelatinisation temperature and higher peak        temperature;    -   (x) reduced viscosity (peak viscosity, pasting temperature,        etc.);    -   (xi) increased molecular weight of amylopectin; and/or    -   (xii) modified % crystallinity % A-type or B-type starch,        relative to a wild-type wheat starch granules or starch.

The present invention further provides a food ingredient that comprisesthe grain, wholemeal, flour, starch granules, or starch as definedherein, for use in the production of foods, for consumption by non-humananimals or preferably humans.

In some embodiments, the food ingredient comprises grain wherein thegrain is kibbled, cracked, par-boiled, rolled, pearled, milled or groundgrain or any combination of these.

The invention also provides food or drink products which comprises afood or drink ingredient at a level of at least 10% on a dry weightbasis, wherein the food ingredient is or comprises the grain, wholemeal,flour, starch granules, or starch as defined herein. Preferably the foodor drink product is packaged ready for sale.

In another embodiment, the invention provides a composition or blendcomprising the grain, wholemeal, flour, wheat starch granules or wheatstarch as defined herein, at a level of at least 10% by weight, andwheat grain having a level of amylose lower than about 50% (w/w) orflour, wholemeal, starch granules or starch obtained therefrom.Preferably, the wheat grain having a level of amylose lower than 50%(w/w) is wild-type wheat grain.

Methods are provided for obtaining or identifying or selecting orproducing a wheat plant that produces grain comprising an amylosecontent of at least 50% (w/w), or at least 60% (w/w), or at least 67%(w/w) as a proportion of the total starch in the grain. The wheat plantmay be identified or selected from a population of multiple candidateplants, such as a mutagenised population or a population of plantsresulting from a crossing process or a back-crossing/breeding process.

In some embodiments, the method comprises: (i) crossing two parentalwheat plants each comprising a loss of function mutation in each of one,two or three SBEIIa or SBEIIb genes selected from the group consistingof SBEIIa-A, SBEIIa-B, SBEIIa-D, SBEIIb-A, SBEIIb-B and SBEIIb-D, or ofmutagenising a parental plant comprising said loss of functionmutations; and (ii) screening plants or grain obtained from the cross ormutagenesis, or progeny plants or grain obtained therefrom, by analysingDNA, RNA, protein, starch granules or starch from the plants or grain,and (iii) selecting a fertile plant that exhibits a level or activity ofSBEII or SBEIIa in its grain that is 2% to 30% the level or activity ofthe respective protein in a wild-type grain. Alternatively, the methodcomprises steps (ii) and (iii) above, with step (i) being optional, suchas when selecting or identifying a plant from a population of multiplecandidate plants.

In some embodiments of the method step (ii) includes screening first,second and/or subsequent generation progeny plants or grain for a lossof function mutation in 5 to 12 alleles of 6 endogenous genes encodingSBEII protein including 4, 5 or 6 SBEIIa alleles, and wherein when thenumber of mutant SBEIIa alleles is 4 then the number of mutant SBEIIballeles is 6, and when the number of mutant SBEIIa alleles is 6 then atleast two such mutants are partial mutations.

In some embodiments, the grain of the selected fertile wheat plant ischaracterised by one or more features as defined herein.

The invention further provides methods of obtaining a hexaploid ortetraploid wheat plant that produces grain comprising an amylose contentof at least 50% (w/w), or at least 60% (w/w) or at least 67% (w/w) as aproportion of the total starch in the grain. In some embodiments, themethod comprises (i) introducing into a wheat cell an exogenous nucleicacid that encodes an RNA which reduces expression of one or more genesencoding total SBEII protein or SBEIIa protein, (ii) regenerating atransgenic wheat plant comprising the exogenous nucleic acid from thecell of step (i), and (iii) screening for and selecting first, second orsubsequent generation progeny of the transgenic wheat plant whichproduce grain having 2% to 30% of the level or activity of total SBEIIor SBEIIa protein in a wild-type plant. Preferably, the RNA molecule isa double-stranded RNA molecule or a micro-RNA precursor molecule, whichis preferably expressed from a chimeric DNA comprising a DNA regionwhich, when transcribed, produces the RNA molecule, operably linked to aheterologous promoter such as an endosperm-specific promoter. Thechimeric DNA may be introduced into a wheat cell which comprises one ormore SBEIIa or SBEIIb mutations, such that the total SBEII activity isreduced in the transgenic plant by a combination of mutation(s) andinhibitory RNA molecule(s).

In some embodiments, grain having 2% to 30% of the level or activity oftotal SBEII or SBEIIa protein in a wild-type plant is indicative that atleast 3 SBEIIa genes or 2 SBEIIa genes and 3 SBEIIb genes of the plantcomprise a loss of function mutation and therefore that grain of theplant comprises more than 50% (w/w), or at least 60% (w/w), or at least67% (w/w) amylose as a proportion of the total starch in the grain.

In some embodiments, the presence of at least a low level of SBEIIaprotein is indicative that the plant is fertile.

In another embodiment, the invention provides a method of screening awheat plant or grain, the method comprising screening a plant or grainfor mutations in a SBEIIa gene or SBEIIa and SBEIIb genes in each of A,B and D genomes of hexaploid wheat or the A and B genomes of tetraploidwheat using one or more of the primers selected from the groupconsisting of SEQ ID NO: 36 to 149.

In another embodiment, the invention provides a method of screening awheat plant or grain, the method comprising (i) determining the level oractivity of SBEIIa and/or SBEIIb relative to the level or activity in awild type or control plant or grain and selecting plant or grain having2% to 30% of the level or activity of total SBEII or SBEIIa protein in awild-type plant.

In yet another embodiment, the invention provides a method of producinga food or a drink comprising (i) obtaining grain of the invention, (ii)processing the grain to produce a food or drink ingredient, and (iii)adding food or drink ingredient from (ii) to another food or drinkingredient, thereby producing the food or drink.

In another aspect, the invention provides a method for improving one ormore parameters of metabolic health, bowel health or cardiovascularhealth in a subject, or of preventing or reducing the severity orincidence of a metabolic disease such as diabetes, bowel disease orcardiovascular disease, comprising providing to the subject the grain,food or drink as defined herein. The invention also provides for the useof the grain, or products derived therefrom, for use in therapy orprophylaxis of the metabolic disease, bowel disease or cardiovasculardisease.

Accordingly, similar aspects of the invention provide the subject grain,food or drink for using in for improving one or more parameters ofmetabolic health, bowel health or cardiovascular health in a subject, orof preventing or reducing the severity or incidence of a metabolicdisease such as diabetes, bowel disease or cardiovascular disease.

Accordingly, similar aspects the invention provide for the use of thesubject grain, food or drink for improving one or more parameters ofmetabolic health, bowel health or cardiovascular health in a subject, orof preventing or reducing the severity or incidence of a metabolicdisease such as diabetes, bowel disease or cardiovascular disease.

Accordingly, in some embodiments the invention provides the food ordrink product as defined herein for use in improving one or moreparameters of metabolic health, bowel health or cardiovascular health,or of preventing or reducing the severity or incidence of metabolic,bowel or cardiovascular disease in a subject.

In another embodiment, the invention provides a method of producinggrain, comprising the steps of i) obtaining a wheat plant that iscapable of producing the grain as defined herein comprising an endospermand a low level or activity of total SBEII protein or SBEIIa proteinthat is 2% to 30% of the level or activity of total SBEII or SBEIIaprotein in a wild-type wheat grain, and wherein the grain comprises anamylose content of at least 50% (w/w), or at least 60% (w/w), or atleast 67% (w/w) as a proportion of the total starch in the grain and,ii) harvesting wheat grain from the plant, and iii) optionally,processing the grain.

In another embodiment, the invention provides a method of producingstarch, comprising the steps of i) obtaining wheat grain as definedherein including comprising an endosperm and a low level or activity oftotal SBEII protein or SBEIIa protein that is 2% to 30% of the level oractivity of total SBEII or SBEIIa protein in a wild-type wheat grain,and wherein the grain comprises an amylose content of at least 50%(w/w), or at least 60% (w/w), or at least 67% (w/w) as a proportion ofthe total starch in the grain, and ii) extracting the starch from thegrain, thereby producing the starch.

The present invention also provides a method of trading wheat grain,comprising obtaining wheat grain of the invention, and trading theobtained wheat grain for pecuniary gain.

In some embodiments, obtaining the wheat grain comprises cultivating orharvesting the wheat grain.

In some embodiments, obtaining the wheat grain comprises harvesting thewheat grain.

In some embodiments, obtaining the wheat grain further comprises storingthe wheat grain.

In some embodiments, obtaining the wheat grain further comprisestransporting the wheat grain to a different location.

The above summary is not and should not be seen in any way as anexhaustive recitation of all embodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation showing an alignment of SBE IIa proteinalignment (AAK26821.1 (SEQ ID NO: 3) is from the D genome, CAR95900.1(SEQ ID NO: 2) from the B genome and CAA72154 (SEQ ID NO: 1) from the Agenome). Dots in the alignment indicate the identical amino acid ispresent as in the uppermost sequence

FIG. 2 is a representation showing an alignment of SBEIIb amino acidsequences encoded by exons 1 to 3 from the A (SEQ ID NO: 4), B (SEQ IDNO: 5) and D (SEQ ID NO: 6) genomes of wheat. Dashes indicate aminoacids are present in the protein but the sequence not known, dots in thealignment indicate the identical amino acid is present as in theuppermost sequence.

FIG. 3 is a representation of an alignment of SBEIIb amino acidsequences. SBEIIb turgidum A/B genome (SEQ ID NO: 150).

FIG. 4 is a graphical representation showing a scatter plot of amylosecontent of transgenic mutant lines (see Example 5).

FIG. 5 is a graphical representation of data showing an amylose modelderived from behaviour of SBEII transgenic lines.

FIG. 6 is a graphical representation of data showing an amylose modelderived from behaviour of SBEII transgenic line.

FIG. 7 is a representation showing an alignment of DNA sequences of theexons 12 to 14 region of homoeologous SBEIIa genes obtained from thewheat variety Chara. The nucleotide sequence for the Chara B genomefragment (SEQ ID NO: 151) is shown in its entirety, while thecorresponding nucleotides for the homoeologous A and D genome fragmentsare shown only where there are polymorphisms. Dots indicate thecorresponding nucleotides are identical to the Chara B genome fragment.Dashes indicate that the corresponding nucleotide is absent from thesequence.

FIG. 8 is a representation showing an alignment of DNA sequences of theintron 3 region of SBEIIa genes obtained from the wheat varieties Suncoand Tasman. The nucleotide sequence for the Tasman D (SEQ ID NO: 152)genome fragment is shown in its entirety, while the correspondingnucleotides for the homoeologous fragments are shown only where thereare polymorphisms. Dots indicate the corresponding nucleotides areidentical to the Tasman D genome fragment. Dashes indicate that thecorresponding nucleotide is absent from the sequence.

FIG. 9 is a representation showing an alignment of DNA sequences of theexon 3 region of homoeologous SBEIIa genes obtained from the wheatvariety Chinese Spring. The nucleotide sequence for the Chinese Spring Dgenome fragment (SEQ ID NO: 153) is shown in its entirety, while thecorresponding nucleotides for the homoeologous A and B genome fragmentsare shown only where there are polymorphisms. Dots indicate thecorresponding nucleotides are identical to the Chinese Spring D genomefragment.

FIG. 10 is a representation showing a DNA sequence of exon 1 region ofSBEIIa gene from the hexaploid wheat variety Chinese Spring (SEQ ID NO:154).

FIG. 11 is a representation showing a PCR amplification of the regionspanning exons 12-14 of SBEIIa genes from CS nullisomic-tetrasomiclines. The line designated BDD is null for A genome, ADD is a null for Bgenome and AAB is a null for D genome.

FIG. 12 is photographic representation of a Western blot showing SBEIIaprotein expression in developing endosperms from the line S28. Proteinextracts from endosperms were assayed by Western blot analysis asdescribed in Example 1, using SBEIIa-specific antibodies. The last laneon the right-hand side shows the bands appearing from wild-typeendosperm (variety NB1). The positions of SBEIIa proteins encoded by theA, B and D genomes are indicated.

FIG. 13 is a plot of mobility ratio of interacting SBEIIa in the absence(m0) and presence (m) of β-limit dextrin in 1-D Native PAGE against theconcentration of β-limit dextrin (S). The dissociation constant (Kd) isderived from the equation m0/m=1+[S]/Kd.

FIG. 14 shows the relationship of amylose content and enzyme resistantstarch in pooled wheat starch samples derived from transgenic wheatlines described in Example 2

FIG. 15 provides scatter plot representations of NIRS-predicted andbiochemical reference values for apparent amylose content in wheatsingle seeds.

FIG. 16 is a graphical representation showing apparent amylose contentdistribution on WM and WMC populations as determined by NIRS.

FIG. 17A and FIG. 17B are graphical representations of data showing theeffect of adding increasing quantities of wheat lines on waterabsorption (a) and Mixograph mixing times (b).

FIG. 18A and FIG. 18B are graphical representations of data illustratingthe effect of adding increasing quantities of high amylose wheat flouron Resistant Starch (a) and predicted GI (b) (HI %) of small scale breadloaves.

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides starch branching enzyme genes characterized fromcereals.

Table 2 provides an amino acid sub-classification.

Table 3 provides exemplary amino acid substitutions.

Table 4 provides genome specific primers for wheat SBEIIa gene.

Table 5 provides nucleotide sequences of genome specific primers forSBEIIa.

Table 6 provides primers designed to amplify parts of the SBEIIa genespecifically from the A genome of wheat.

Table 7 provides primers designed to amplify parts of the SBEIIa genespecifically from the B genome of wheat.

Table 8 provides primers designed to amplify parts of the SBEIIa genespecifically from the D genome of wheat.

Table 9 provides genome specific primers for wheat SBEIIb gene.

Table 10 provides nucleotide sequences of genome specific primers forSBEIIb.

Table 11 provides total SBEII and SBEIIa and SBEIIb expression andamylose content of RNAi lines of wheat as described in Example 4.

Table 12 provides a list of microsatellite markers tested in the mutantsas described in Example 5.

Table 13 provides mutants identified from HIB population andmicrosatellite mapping data as described in Example 5.

Table 14 provides a description of double null mutants of SBEIIidentified as described in Example 5.

Table 15 provides a description of crosses performed between double andsingle null mutants as described in Example 5.

Table 16 provides tabulation of amylose content in grain starch oftriple nulls mutants as described in Example 5.

Table 17 provides fertility observations on F2 progeny plants.

Table 18 provides SBEII allelic composition and amylose proportion datafor double nulls identified.

Table 19 provides details of further crosses between single and doublenull mutants.

Table 20 provides observed frequency of genotypes of normallygerminating grain from an A2B2D2 cross. Numbers in parentheses indicatethe expected frequency based on Mendelian segregation.

Table 21 provides further crosses between single and double nullmutants.

Table 22 provides putative double and triple null mutants in SBEIIagenes identified in an initial screen using dominant markers.

Table 23 provides starch characterisation of grain starch fromtransgenic wheat lines.

Table 24 provides molecular weight distribution of starch fractions fromwheat transgenic lines.

Table 25 provides RVA parameters of hp5′-SBEIIa transgenic wheat starch.

Table 26 provides DSC parameters of gelatinisation peak of hp5′-SBEIIatransgenic wheat starch compared to the control NB1.

Table 27 provides RS content in rolled and flaked grain products.

Table 28 provides resistant starch content in food products at varyinglevel of incorporation of high amylose wheat (HAW).

Table 29 provides genome-specific primers referred to in Example 18.

DETAILED DESCRIPTION

The present invention is based in part on the observations made in theexperiments described herein that wheat plants completely lacking SBEIIaactivity throughout the plant could not be recovered in crosses designedto produce them, indeed the complete lack of SBEIIa was concluded to belethal to seed development and/or fertility. This was surprising sinceprevious studies have shown that single null mutants in SBEIIa couldreadily be obtained in wheat and were fertile. Moreover, it was observedthat the minimum level of SBEIIa activity that needed to be retained inthe wheat plant to produce normal, viable seed was about 2% of thewild-type level.

It was also observed that mutant plants and grain comprising at leastone point mutation in an SBEIIa gene were favoured over plants and grainwhich had deletions in each of the SBEIIa genes for combining mutantSBEIIa genes, in particular to obtain phenotypically normal, male andfemale fertile plants and grain which germinated at rates similar towild-type grain. One possible explanation of this observation was thatdeletions tend to remove important genetic elements adjacent to theSBEIIa genes.

It was also observed that to obtain an amylose content of at least 50%(w/w) in the grain starch, at which level the amount of resistant starchand associated health benefits were increased substantially, the totalSBEII activity and particularly the SBEIIa activity in the grain neededto be reduced to below 30% of the wild-type level.

Furthermore, it was determined that, in hexaploid wheat, reducing thelevel and/or activity of SBEII protein from each of three homoeologousSBEIIa genes or from at least two homoeologous SBEIIa genes and two orthree homoeologous SBEIIb genes leads to a substantial non-linearincrease in the proportion of amylose in starch of the wheat endospermcompared to plants having null mutation in two homoeologous SBEIIagenes. This non-linear relationship between amylose content and SBEIIlevels in grain of hexaploid wheat is illustrated graphically in FIGS. 5and 6.

By studying partial and complete loss of function mutations incombinations of SBEIIa and/or SBEIIb alleles from A, B and D genomes,the role of multiple SBEII genes in modulating starch characteristicshas been established. Specifically, the number of mutant alleles andcombinations of mutant alleles required to obtain fertile wheat plantshaving very high levels of amylose has been investigated and determined.

The synthesis of starch in the endosperm of higher plants includingwheat is carried out by a suite of enzymes that catalyse four key steps.Firstly, ADP-glucose pyrophosphorylase (EC 2.7.7.27) activates themonomer precursor of starch through the synthesis of ADP-glucose fromG-1-P and ATP. Secondly, the activated glucosyl donor, ADP-glucose, istransferred to the non-reducing end of a pre-existing α(1-4) linkage bystarch synthases (EC 2.4.1.24). Thirdly, starch branching enzymesintroduce branch points through the cleavage of a region of α(1-4)linked glucan followed by transfer of the cleaved chain to an acceptorchain, forming a new α(1-6) linkage. Starch branching enzymes are theonly enzymes that can introduce the α(1-6) linkages into α-polyglucansand therefore play an essential role in the formation of amylopectin.Fourthly, starch debranching enzymes (EC 2.4.4.18) remove some of thebranch linkages.

Starch is the major storage carbohydrate in plants such as cereals,including wheat. Starch is synthesized in the amyloplasts and formed andstored in granules in the developing storage organ such as grain; it isreferred to herein as “storage starch” or “grain starch”. In cerealgrains, the vast majority of the storage starch is deposited in theendosperm. “Starch” is defined herein as polysaccharide composed ofglucopyranose units polymerized through a combination of both α(1-4) andα(1-6) linkages. The polydisperse molecules of starch are classified asbelonging to two component fractions, known as amylose and amylopectin,on the basis of their degree of polymerization (DP) and the ratio ofα(1-6) to α(1-4) linkages. Grain starch from wild-type cereal plants,including from wheat, comprises about 20%-30% of amylose and about70%-80% of amylopectin.

“Amylose” is defined herein as including essentially linear molecules ofα(1,4) linked glucosidic (glucopyranose) units, sometimes referred to as“true amylose”, and amylose-like long-chain starch which is sometimesreferred to as “intermediate material” or “amylose-like amylopectin”which appears as iodine-binding material in an iodometric assay alongwith true amylose (Takeda et al., 1993b; Fergason, 1994). Typically, thelinear molecules in true amylose have a DP of between 500 and 5000 andcontain less than 1% α(1-6) linkages. Recent studies have shown thatabout 0.1% of α(1-6)-glycosidic branching sites may occur in amylose,therefore it is described as “essentially linear”. In contrast,amylopectin is a much larger molecule with a DP ranging from 5000 to50,000 and contains 4-5% α(1-6) linkages. Amylopectin molecules aretherefore more highly branched. Amylose has a helical conformation witha molecular weight of about 10⁴ to about 10⁶ Daltons while amylopectinhas a molecular weight of about 10⁷ to about 10⁸ Daltons. These twotypes of starch can readily be distinguished or separated by methodswell known in the art.

The proportion of amylose in the starch as defined herein is on aweight/weight (w/w) basis, i.e. the weight of amylose as a percentage ofthe weight of total starch extractable from the grain, with respect tothe starch prior to any fractionation into amylose and amylopectinfractions. The terms “proportion of amylose in the starch” and “amylosecontent” when used herein in the context of the grain, flour or otherproduct of the invention are essentially interchangeable terms. Amylosecontent may be determined by any of the methods known in the artincluding size exclusion high-performance liquid chromatography (HPLC),for example in 90% (w/v) DMSO, concanavalin A methods (Megazyme Int,Ireland), or preferably by an iodometric method, for example asdescribed in Example 1. The HPLC method may involve debranching of thestarch (Batey and Curtin, 1996) or not involve debranching. It will beappreciated that methods such as the HPLC method of Batey and Curtin,1996 which assay only the “true amylose” may underestimate the amylosecontent as defined herein. Methods such as HPLC or gel permeationchromatography depend on fractionation of the starch into the amyloseand amylopectin fractions, while iodometric methods depend ondifferential iodine binding and therefore do not require fractionation.

From the grain weight and amylose content, the amount of amylosedeposited per grain can be calculated and compared for test and controllines.

Starch is initially synthesized and accumulated in the leaves and othergreen tissues of a plant as a product of photosynthesis. This starch isreferred to herein as “transitory starch” or the like because, incontrast to seed or tuber starch, it accumulates in the plastids of thephotosynthetic tissues during the day and is degraded at least duringthe night. At night, transitory starch is hydrolysed to sugars which aretransported, primarily as sucrose, from the source tissues to sinktissues for use in growth of the plant, as an energy source formetabolism or for storage in tissues as storage starch.

As used herein, “starch synthase” means an enzyme that transfersADP-glucose to the non-reducing end of a pre-existing α1-4 linkages.Four classes of starch synthase are found in the cereal endosperm, anisoform exclusively localised within the starch granule, granule-boundstarch synthase (GBSS), two forms that are partitioned between thegranule and the soluble fraction (SSI, Li et al., 1999a; SSII, Li etal., 1999b) and a fourth form that is entirely located in the solublefraction, SSIII (Cao et al., 2000; Li et al., 1999b; Li et al., 2000).GBSS has been shown to be essential for amylose synthesis (Shure et al.,1983), and mutations in SSII and SSIII have been shown to alteramylopectin structure (Gao et al., 1998; Craig et al, 1998). Mutants incereals which lack GBSS also lack true amylose and so accumulate onlyamylopectin; these are commonly referred to as “waxy” mutants. Nomutations defining a role for SSI activity have been described.Amyloepectin synthesis is more complex than amylose synthesis, requiringa combination of starch synthases other than GBSS, multiple starchbranching enzymes and debranching enzyme.

As used herein, “debranching enzyme” means an enzyme that removes someof the branches of amylopectin formed by starch branching enzymes. Twotypes of debranching enzymes are present in higher plants and aredefined on the basis of their substrate specificities, isoamylase typedebranching enzymes, and pullulanase type debranching enzymes (Myers etal., 2000). Sugary-1 mutations in maize and rice are associated withdeficiency of both debranching enzymes (James et al., 1995; Kubo et al.,1999) however the causal mutation maps to the same location as theisoamylase-type debranching enzyme gene.

Examples of genes encoding starch branching enzymes from cerealsincluding wheat are given in Table 1. As used herein, “starch branchingenzyme” means an enzyme that introduces α-1,6 glycosidic bonds betweenchains of glucose residues (EC 2.4.1.18). Three forms of starchbranching enzyme are expressed in cereals such as rice, maize, barleyand wheat, including in the developing cereal endosperm, namely starchbranching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa) andstarch branching enzyme IIb (SBEIIb) (Hedman and Boyer, 1982; Boyer andPreiss, 1978; Mizuno et al., 1992, Sun et al., 1997). Genomic and cDNAsequences for genes encoding these enzymes have been characterized forrice, barley and wheat (Table 1). Sequence alignment reveals a highdegree of sequence similarity at both the nucleotide and amino acidlevels, but also the sequence differences and allows the grouping intothe SBEI, SBEIIa and SBEIIb classes. SBEIIa and SBEIIb from any onespecies generally exhibit around 80% amino acid sequence identity toeach other, particularly in the central regions of the genes. SBEIIa andSBEIIb may also be distinguished by their expression patterns, but thisdiffers in different species. In maize, SBEIIb is most highly expressedin endosperm while SBEIIa is present in every tissue of the plant. Inbarley, both SBEIIa and SBEIIb are present in about equal amounts in theendosperm, while in wheat endoperm, SBEIIa is expressed about 4-foldmore highly than SBEIIb. Therefore, the cereal species show significantdifferences in SBEIIa and SBEIIb expression, and conclusions drawn inone species cannot readily be applied to another species. In wheat,SBEIIa and SBEIIb proteins are different in size (see below) and this isa convenient way to distinguish them. Specific antibodies may also beused to distinguish them.

In maize, high amylose phenotypes have been shown to result from lesionsin the SBEIIb gene, also known as the amylose extender (ae) gene (Boyerand Preiss, 1981, Mizuno et al., 1993; Nishi et al., 2001). In theseSBEIIb mutants, endosperm starch grains showed an abnormal morphology,amylose content was significantly elevated, the branch frequency of theresidual amylopectin was reduced and the proportion of short chains(<DP17, especially DP8-12) was lower. Moreover, the gelatinisationtemperature of the starch was increased. In addition, there was asignificant pool of material that was defined as “intermediate” betweenamylose and amylopectin (Boyer et al., 1980; Takeda, et al., 1993b). Incontrast, maize plants mutant in the SBEIIa gene due to a mutator (Mu)insertional element and consequently lacking in SBEIIa proteinexpression were indistinguishable from wild-type plants in the branchingof endosperm starch (Blauth et al., 2001), although they were altered inleaf starch. Similarly, rice plants deficient in SBEIIa activityexhibited no significant change in the amylopectin chain profile inendosperm (Nakamura, 2002), while mutants in SBEIIb showed a modestincrease in amylose levels, up to about 35% in indica backgrounds and upto 25-30% in a japonica background (Mizuno et al., 1993; Nishi et al.,2001). In both maize and rice, the SBEIIa and SBEIIb genes are notlinked in the genome. In barley, a gene silencing construct whichreduced both SBEIIa and SBEIIb expression in endosperm was used togenerate high amylose barley grain (Regina et al., 2010).

In developing wheat endosperm, SBEI (Morell et al., 1997) is foundexclusively in the soluble fraction (amyloplast stroma), while SBEIIaand SBEIIb are found in both soluble and starch-granule associatedfractions in endosperm (Rahman et al., 1995). In wheat, apparent geneduplication events have increased the number of SBEI genes in eachgenome (Rahman et al., 1999). The elimination of greater than 97% of theSBEI activity by combining mutations in the highest expressing forms ofthe SBEI genes from the A, B and D genomes had no measurable impact onstarch structure or functionality (Regina et al., 2004). In contrast,reduction of SBEIIa expression by a gene silencing construct in wheatresulted in high amylose levels (>70%), while a corresponding constructthat reduced SBEIIb expression but not SBEIIa had minimal effect (Reginaet al., 2006).

Starch branching enzyme (SBE) activity may be measured by enzyme assay,for example by the phosphorylase stimulation assay (Boyer and Preiss,1978). This assay measures the stimulation by SBE of the incorporationof glucose 1-phosphate into methanol-insoluble polymer (α-D-glucan) byphosphorylase A. SBE activity can be measured by the iodine stain assay,which measures the decrease in the absorbency of a glucan-polyiodinecomplex resulting from branching of glucan polymers. SBE activity canalso be assayed by the branch linkage assay which measures thegeneration of reducing ends from reduced amylose as substrate, followingisoamylose digestion (Takeda et al., 1993a). Preferably, the activity ismeasured in the absence of SBEI activity. Isoforms of SBE show differentsubstrate specificities, for example SBEI exhibits higher activity inbranching amylose, while SBEIIa and SBEIIb show higher rates ofbranching with an amylopectin substrate. The isoforms may also bedistinguished on the basis of the length of the glucan chain that istransferred. SBE protein may also be measured by using specificantibodies such as those described herein. The SBEII activity may bemeasured during grain development in the developing endosperm.Alternatively, SBEII levels are measured in the mature grain where theprotein is still present and can be assayed by immunological methods.

In some embodiments, the level or activity of SBEII or SBEIIa may beassessed by assessing transcript levels such as by Northern or RT-PCRanalysis. In a preferred method, the amount of SBEIIa protein in grainor developing endosperm is measured by separating the proteins inextracts of the grain/endosperm on gels by electrophoresis, thentransferring the proteins to a membrane by Western blotting, followed byquantitative detection of the protein on the membrane using specificantibodies (“Western blot analysis”). This is exemplified in Example 11.

As shown herein, developing hexaploid wheat endosperm expresses SBEIIaand SBEIIb from each of the A, B and D genomes. Tetraploid wheatexpresses SBEIIa and SBEIIb from each of the A and B genomes. As usedherein, “SBEIIa expressed from the A genome” or “SBEIIa-A” means astarch branching enzyme whose amino acid sequence is set forth in SEQ IDNO: 1 or which is at least 99% identical to the amino acid sequence setforth in SEQ ID NO: 1 or comprising such a sequence. The amino acidsequence of SEQ ID NO: 1 (Genbank Accession No. CAA72154) corresponds toan SBEIIa expressed from the A genome of wheat, which is used herein asthe reference sequence for wild-type SBEIIa-A. The protein of SEQ ID NO:1 is 823 amino acids long. Active variants of this enzyme exist inwheat, for example in cultivar Cheyenne, see Accession No. AF286319which is 99.88% (822/823) identical to SEQ ID NO. 1. Such variants areincluded in “SBEIIa-A” provided they have essentially wild-type starchbranching enzyme activity as for SEQ ID NO: 1.

As used herein, “SBEIIa expressed from the B genome” or “SBEIIa-B” meansa starch branching enzyme whose amino acid sequence is set forth in SEQID NO: 2 or which is at least 99% identical to the amino acid sequenceset forth in SEQ ID NO: 2 or comprising such a sequence. The amino acidsequence of SEQ ID NO: 2 (Genbank Accession No. CAR95900) corresponds tothe SBEIIa expressed from the B genome of wheat variety Chinese Spring,which is used herein as the reference sequence for wild-type SBEIIa-B.The protein of SEQ ID NO: 2 is 823 amino acids long. Active variants ofthis enzyme may exist in wheat and are included in SBEIIa-B providedthey have essentially wild-type starch branching enzyme activity as forSEQ ID NO: 2. SEQ ID NO: 2 is 98.42% (811/824) identical to SEQ IDNO: 1. The alignment of the amino acid sequences in FIG. 1 shows theamino acid differences which may be used to distinguish the proteins orto classify variants as SBEIIa-A or SBEIIa-B.

As used herein, “SBEIIa expressed from the D genome” or “SBEIIa-D” meansa starch branching enzyme whose amino acid sequence is set forth in SEQID NO: 3 or which is at least 98% identical to the amino acid sequenceset forth in SEQ ID NO: 3 or comprising such a sequence. The amino acidsequence of SEQ ID NO: 3 (Genbank Accession No. AAK26821) corresponds tothe SBEIIa expressed from the D genome in A. tauschii, a likelyprogenitor of the D genome of hexaploid wheat, which is used herein asthe reference sequence for wild-type SBEIIa-D. The protein of SEQ ID NO:3 is 819 amino acids long. Active variants of this enzyme may exist inwheat and are included in SBEIIa-D provided they have essentiallywild-type starch branching enzyme activity as for SEQ ID NO: 3. SEQ IDNO: 3 is 97.57% (803/823) identical to SEQ ID NO: 1 and 97.81% (805/823)identical to SEQ ID NO: 2. The alignment of the amino acid sequences inFIG. 1 shows amino acid differences which may be used to distinguish theproteins or to classify variants as SBEIIa-A, SBEIIa-B or SBEIIa-D.

When comparing amino acid sequences to determine the percentage identityin this context, for example by Blastn, the full length sequences shouldbe compared, and gaps in a sequence counted as amino acid differences.

As used herein, an “SBEIIa protein” includes protein variants which havereduced or no starch branching enzyme activity, as well as the proteinshaving essentially wild-type enzyme activity. It is also understood thatSBEIIa proteins may be present in grain, particularly dormant grain ascommonly harvested commercially, but in an inactive state because of thephysiological conditions in the grain. Such proteins are included in“SBEIIa proteins” as used herein. The SBEIIa proteins may beenzymatically active during only part of grain development, inparticular in developing endosperm when storage starch is typicallydeposited, but in inactive state otherwise. Such SBEIIa protein may bedetected and quantitated readily using immunological methods such asWestern blot analysis. An “SBEIIb protein” as used herein has ananalogous meaning.

As used herein, “SBEIIb expressed from the A genome” or “SBEIIb-A” meansa starch branching enzyme comprising the amino acid sequence set forthin SEQ ID NO: 4 or which is at least 98% identical to the amino acidsequence set forth in SEQ ID NO: 4 or comprising such a sequence. Theamino acid sequence of SEQ ID NO: 4 corresponds to the amino terminalsequence of SBEIIb expressed from the A genome of wheat, which is usedherein as the reference sequence for wild-type SBEIIb-A.

As used herein, “SBEIIb expressed from the B genome” or “SBEIIb-B” meansa starch branching enzyme comprising the amino acid sequence set forthin SEQ ID NO: 5 or which is at least 98% identical to the amino acidsequence set forth in SEQ ID NO: 5 or comprising such a sequence. Theamino acid sequence SEQ ID NO: 5, which is used herein as the referencesequence for wild-type SBEIIb-B, is a partial amino acid sequenceencoded by exons 2-3 of the SBEIIb-B gene in wheat. A variant SBEIIb-Bsequence is the amino acid sequence encoded by the nucleotide sequenceof Accession No. AK335378 isolated from cv. Chinese Spring.

As used herein, “SBEIIb expressed from the D genome” or “SBEIIb-D” meansa starch branching enzyme whose amino acid sequence is set forth in SEQID NO: 6 or which is at least 98% identical to the amino acid sequenceset forth in SEQ ID NO: 6 or comprising such a sequence. The amino acidsequence of SEQ ID NO: 6 (Genbank Accession No. AAW80631) corresponds tothe SBEIIb expressed from the D genome of A. tauschii, a likelyprogenitor of the D genome of hexaploid wheat, and is used herein as thereference sequence for wild-type SBEIIa-D. Active variants of thisenzyme exist in wheat and are included in SBEIIb-D provided they haveessentially wild-type starch branching enzyme activity as for SEQ ID NO:6. For example, SEQ ID NO: 4 of US patent application publication No.20050074891, beginning at the first methionine, shows the amino acidsequence of a SBEIIb-D protein which is 99.5% identical to SEQ ID NO: 6in this application. The alignment of the amino acid sequences in FIG. 2shows amino acid differences which may be used to distinguish SBEIIbproteins or to classify variants as SBEIIb-A, SBEIIb-B or SBEIIb-D.

Thus, “wild-type” as used herein when referring to SBEIIa-A means astarch branching enzyme whose amino acid sequence is set forth in SEQ IDNO: 1; “wild-type” as used herein when referring to SBEIIa-B means astarch branching enzyme whose amino acid sequence is set forth in SEQ IDNO: 2; “wild-type” as used herein when referring to SBEIIa-D means astarch branching enzyme whose amino acid sequence is set forth in SEQ IDNO: 3; “wild-type” as used herein when referring to SBEIIb-A means astarch branching enzyme whose amino acid sequence is set forth in SEQ IDNO: 4; “wild-type” as used herein when referring to SBEIIb-B means astarch branching enzyme whose amino acid sequence is set forth in SEQ IDNO: 5; and, “wild-type” as used herein when referring to SBEIIb-D meansa starch branching enzyme whose amino acid sequence is set forth in SEQID NO: 6.

As used herein, the terms “wheat SBEIIa gene” and “wheat SBEIIb gene”refer to the genes that encode functional SBEIIa or SBEIIb enzymes,respectively, in wheat, including homologous genes present in otherwheat varieties, and also mutant forms of the genes which encode enzymeswith reduced activity or undetectable activity. These include, but arenot limited to, the wheat SBEII genes which have been cloned, includingthe genomic and cDNA sequences listed in Table 1. The genes as usedherein encompasses mutant forms which do not encode any proteins at all,in which case the mutant forms represent null alleles of the genes.

An “endogenous SBEII gene” refers to an SBEII gene which is in itsnative location in the wheat genome, including wild-type and mutantforms. In contrast, the terms “isolated SBEII gene” and “exogenous SBEIIgene” refer to an SBEII gene which is not in its native location, forexample having been cloned, synthesized, comprised in a vector or in theform of a transgene in a cell, preferably as transgene in a transgenicwheat plant. The SBEII gene in this context may be any of the specificforms as described as follows.

As used herein, “the SBEIIa gene on the A genome of wheat” or “SBEIIa-Agene” means any polynucleotide which encodes SBEIIa-A as defined hereinor which is derived from a polynucleotide which encodes SBEIIa-A,including naturally occurring polynucleotides, sequence variants orsynthetic polynucleotides, including “wild-type SBEIIa-A gene(s)” whichencode an SBEIIa-A with essentially wild-type activity, and “mutantSBEIIa-A gene(s)” which do not encode an SBEIIa-A with essentiallywild-type activity but are recognizably derived from a wild-typeSBEIIa-A gene. Comparison of the nucleotide sequence of a mutant form ofan SBEII gene with a suite of wild-type SBEII genes is used to determinewhich of the SBEII genes it is derived from and so to classify it. Forexample, a mutant SBEII gene is considered to be a mutant SBEIIa-A geneif its nucleotide sequence is more closely related, i.e. having a higherdegree of sequence identity, to a wild-type SBEIIa-A gene than to anyother SBEII gene. A mutant SBEIIa-A gene encodes a SBE with reducedstarch branching enzyme activity (partial mutant), or a protein whichlacks SBE activity or no protein at all (null mutant gene). An exemplarynucleotide sequence of a cDNA corresponding to a SBEIIa-A gene is givenin Genbank Accession No. Y11282. Sequences of parts of SBEIIa-A genesare also given herein as referred to in FIGS. 7, 8, 9 and 10 and SEQ IDNOs 13, 14 and 15.

As used herein, the terms “SBEIIa expressed from the B genome” or“SBEIIa-B”, “SBEIIa expressed from the D genome” or “SBEIIa-D”, “SBEIIbexpressed from the A genome” or “SBEIIb-A”, “SBEIIb expressed from the Bgenome” or “SBEIIb-B” and “SBEIIb expressed from the D genome” or“SBEIIb-D” have corresponding meanings to that for SBEIIa-A in theprevious paragraph.

Illustrative partial SBEIIb-A, SBEIIb-B and SBEIIb-D protein sequencesare provided in FIG. 2. Illustrative SBEIIb-A amino acid sequences areset out in SEQ ID NO: 1 and SEQ ID NO: 4 (amino terminal sequenceencoded by exon 1-3). Illustrative SBEIIb-B amino acid sequences are setout in SEQ ID NO: 2 and SEQ ID NO: 5. Illustrative SBEIIb-D amino acidsequences are set out in SEQ ID NO: 3 and SEQ ID NO: 6 and SEQ ID NO: 9.

The SBEII genes as defined above include any regulatory sequences thatare 5′ or 3′ of the transcribed region, including the promoter region,that regulate the expression of the associated transcribed region, andintrons within the transcribed regions.

It would be understood that there is natural variation in the sequencesof SBEIIa and SBEIIb genes from different wheat varieties. Thehomologous genes are readily recognizable by the skilled artisan on thebasis of sequence identity. The degree of sequence identity betweenhomologous SBEIIa genes or the proteins is thought to be at least 90%,similarly for SBEIIb genes or proteins. Wheat SBEIIa genes are about 80%identical in sequence to wheat SBEIIb genes. The encoded proteins arealso about 80% identical in sequence.

An allele is a variant of a gene at a single genetic locus. A diploidorganism has two sets of chromosomes. Each chromosome has one copy ofeach gene (one allele). If both alleles are the same the organism ishomozygous with respect to that gene, if the alleles are different, theorganism is heterozygous with respect to that gene. The interactionbetween alleles at a locus is generally described as dominant orrecessive. A loss of function mutation is a mutation in an alleleleading to no or a reduced detectable level or activity of SBEII, SBEIIaor SBEIIb enzyme in the grain. The mutation may mean, for example, thatno or less RNA is transcribed from the gene comprising the mutation orthat the protein produced has no or reduced activity. Alleles that donot encode or are not capable of leading to the production any activeenzyme are null alleles. A loss of function mutation, which includes apartial loss of function mutation in an allele, means a mutation in theallele leading to a reduced level or activity of SBEII, SBEIIa or SBEIIbenzyme in the grain. The mutation in the allele may mean, for example,that less protein having wild-type or reduced activity is translated orthat wild-type or reduced levels of transcription are followed bytranslation of an enzyme with reduced enzyme activity. A “reduced”amount or level of protein means reduced relative to the amount or levelproduced by the corresponding wild-type allele. A “reduced” activitymeans reduced relative to the corresponding wild-type SBEII, SBEIIa orSBEIIb enzyme. Different alleles in the embryo may have the same or adifferent mutation and different alleles may be combined using methodsknown in the art. In some embodiments, the amount of SBEIIa protein orSBEIIb protein is reduced because there is less transcription ortranslation of the SBEIIa gene or SBEIIb gene, respectively. In someembodiments, the amount by weight of SBEIIa protein or SBEIIb protein isreduced even though there is a wild-type number of SBEIIa proteinmolecules or SBEIIb protein molecules in the grain, because some of theproteins produced are shorter than wild-type SBEIIa protein or SBEIIbprotein, e.g. the mutant SBEIIa protein or SBEIIb protein is truncateddue to a premature translation termination signal.

Representative starch biosynthesis genes that have been cloned fromcereals are listed in Table 1.

As used herein, “two identical alleles of an SBEIIa-A gene”, means thatthe two alleles of the SBEIIa-A gene are identical to each other; “twoidentical alleles of an SBEIIa-B gene”, means that the two alleles ofthe SBEIIa-B gene are identical to each other; “two identical alleles ofan SBEIIa-D gene”, means that the two alleles of the SBEIIa-D gene areidentical to each other; “two identical alleles of an SBEIIb-A gene”,means that the two alleles of the SBEIIb-A gene are identical to eachother; “two identical alleles of an SBEIIb-B gene”, means that the twoalleles of the SBEIIb-B gene are identical to each other; and, “twoidentical alleles of an SBEIIb-D gene”, means that the two alleles ofthe SBEIIb-D gene are identical to each other.

The wheat plants of the invention can be produced and identified aftermutagenesis. This may provide a wheat plant which is non-transgenic,which is desirable in some markets, or which is free of any exogenousnucleic acid molecule which reduces expression of an SBEIIa gene. Mutantwheat plants having a mutation in a single SBEII gene which can becombined by crossing and selection with other SBEII mutations togenerate the wheat plants of the invention can be either synthetic, forexample, by performing site-directed mutagenesis on the nucleic acid, orinduced by mutagenic treatment, or may be naturally occurring, i.e.isolated from a natural source. Generally, a progenitor plant cell,tissue, seed or plant may be subjected to mutagenesis to produce singleor multiple mutations, such as nucleotide substitutions, deletions,additions and/or codon modification. Preferred wheat plants and grain ofthe invention comprise at least one introduced SBEII mutation, morepreferably two or more introduced SBEII mutations, and may comprise nomutations from a natural source i.e. all of the mutant SBEIIa and SBEIIballeles in the plant were obtained by synthetic means or by mutagenictreatment.

Mutagenesis can be achieved by chemical or radiation means, for exampleEMS or sodium azide (Zwar and Chandler, 1995) treatment of seed, orgamma irradiation, well know in the art. Chemical mutagenesis tends tofavour nucleotide substitutions rather than deletions. Heavy ion beam(HIB) irradiation is known as an effective technique for mutationbreeding to produce new plant cultivars, see for example Hayashi et al.,2007 and Kazama et al, 2008. Ion beam irradiation has two physicalfactors, the dose (gy) and LET (linear energy transfer, keV/um) forbiological effects that determine the amount of DNA damage and the sizeof DNA deletion, and these can be adjusted according to the desiredextent of mutagenesis. HIB generates a collection of mutants, many ofthem comprising deletions that may be screened for mutations in specificSBEII genes as shown in the Examples. Mutants which are identified maybe backcrossed with non-mutated wheat plants as recurrent parents inorder to remove and therefore reduce the effect of unlinked mutations inthe mutagenised genome, see Example 9.

Biological agents useful in producing site-specific mutants includeenzymes that include double stranded breaks in DNA that stimulateendogenous repair mechanisms. These include endonucleases, zinc fingernucleases, transposases and site-specific recombinases. Zinc fingernucleases (ZFNs), for example, facilitate site-specific cleavage withina genome allowing endogenous or other end-joining repair mechanisms tointroduce deletions or insertions to repair the gap. Zinc fingernuclease technology is reviewed in Le Provost et al., 2009, See alsoDurai et al., 2005 and Liu et al., 2010. Isolation of mutants may beachieved by screening mutagenised plants or seed.

For example, a mutagenized population of wheat may be screened directlyfor the SBEIIa and/or SBEIIb genotype or indirectly by screening for aphenotype that results from mutations in the SBEII genes. Screeningdirectly for the genotype preferably includes assaying for the presenceof mutations in the SBEII genes, which may be observed in PCR assays bythe absence of specific SBEIIa or SBEIIb markers as expected when someof the genes are deleted, or heteroduplex based assays as in Tilling.Screening for the phenotype may comprise screening for a loss orreduction in amount of one or more SBEIIa or SBEIIb proteins by ELISA oraffinity chromatography, or increased amylose content in the grainstarch. In hexaploid wheat, screening is preferably done in a genotypethat already lacks one or two of the SBEII activities, for example in awheat plant already mutant in the SBEIIa or SBEIIb genes on two of thethree genomes, so that a mutant further lacking the functional activityis sought. In tetraploid wheat, screening is preferably done in agenotype that already lacks one SBEII activity, on either the A or Bgenome, and identifying a mutant which is reduced in the SBEII from thesecond genome. Affinity chromatography may be carried out asdemonstrated in Example 11. Large populations of mutagenised seeds(thousands or tens of thousands of seeds) may be screened for highamylose phenotypes using near infra-red spectroscopy (NIR) asdemonstrated in Example 10. Using NIR, a sub-population enriched forhigh amylose candidates was obtainable. By these means, high throughputscreening is readily achievable and allows the isolation of mutants at afrequency of approximately one per several hundred seeds.

Plants and seeds of the invention can be produced using the processknown as TILLING (Targeting Induced Local Lesions IN Genomes), in thatone or more of the mutations in the wheat plants or grain may beproduced by this method. In a first step, introduced mutations such asnovel single base pair changes are induced in a population of plants bytreating seeds or pollen with a chemical or radiation mutagen, and thenadvancing plants to a generation where mutations will be stablyinherited, typically an M2 generation where homozygotes may beidentified. DNA is extracted, and seeds are stored from all members ofthe population to create a resource that can be accessed repeatedly overtime. For a TILLING assay, PCR primers are designed to specificallyamplify a single gene target of interest. Next, dye-labeled primers canbe used to amplify PCR products from pooled DNA of multiple individuals.These PCR products are denatured and reannealed to allow the formationof mismatched base pairs. Mismatches, or heteroduplexes, represent bothnaturally occurring single nucleotide polymorphisms (SNPs) (i.e.,several plants from the population are likely to carry the samepolymorphism) and induced SNPs (i.e., only rare individual plants arelikely to display the mutation). After heteroduplex formation, the useof an endonuclease, such as Cel I, that recognizes and cleavesmismatched DNA is the key to discovering novel SNPs within a TILLINGpopulation.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling and amplifying 1.4 kb fragments with 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique. TILLING is further described in Slade andKnauf, 2005, and Henikoff et al., 2004.

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).Plates containing arrayed ecotypic DNA can be screened rather than poolsof DNA from mutagenized plants. Because detection is on gels with nearlybase pair resolution and background patterns are uniform across lanes,bands that are of identical size can be matched, thus discovering andgenotyping mutations in a single step. In this way, sequencing of themutant gene is simple and efficient.

Identified mutations may then be introduced into desirable geneticbackgrounds by crossing the mutant with a plant of the desired geneticbackground and performing a suitable number of backcrosses to cross outthe originally undesired parent background.

In the context of this application, an “induced mutation” or “introducedmutation” is an artificially induced genetic variation which may be theresult of chemical, radiation or biologically-based mutagenesis, forexample transposon or T-DNA insertion. Preferred mutations are nullmutations such as nonsense mutations, frameshift mutations, deletions,insertional mutations or splice-site variants which completelyinactivate the gene. Other preferred mutations are partial mutationswhich retain some SBEII activity, but less than wild-type levels of theenzyme. Nucleotide insertional derivatives include 5′ and 3′ terminalfusions as well as intra-sequence insertions of single or multiplenucleotides. Insertional nucleotide sequence variants are those in whichone or more nucleotides are introduced into a site in the nucleotidesequence, either at a predetermined site as is possible with zinc fingernucleases (ZFN) or other homologous recombination methods, or by randominsertion with suitable screening of the resulting product. Deletionalvariants are characterised by the removal of one or more nucleotidesfrom the sequence. Preferably, a mutant gene has only a single insertionor deletion of a sequence of nucleotides relative to the wild-type gene.The deletion may be extensive enough to include one or more exons orintrons, both exons and introns, an intron-exon boundary, a part of thepromoter, the translational start site, or even the entire gene.Deletions may extend far enough to include at least part of, or thewhole of, both the SBEIIa and SBEIIb genes on the A, B or D genome,based on the close genetic linkage of the two genes. Insertions ordeletions within the exons of the protein coding region of a gene whichinsert or delete a number of nucleotides which is not an exact multipleof three, thereby causing a change in the reading frame duringtranslation, almost always abolish activity of the mutant genecomprising such insertion or deletion.

Substitutional nucleotide variants are those in which at least onenucleotide in the sequence has been removed and a different nucleotideinserted in its place. The preferred number of nucleotides affected bysubstitutions in a mutant gene relative to the wild-type gene is amaximum of ten nucleotides, more preferably a maximum of 9, 8, 7, 6, 5,4, 3, or 2, or most preferably only one nucleotide. Substitutions may be“silent” in that the substitution does not change the amino acid definedby the codon. Nucleotide substitutions may reduce the translationefficiency and thereby reduce the SBEII expression level, for example byreducing the mRNA stability or, if near an exon-intron splice boundary,alter the splicing efficiency. Silent substitutions that do not alterthe translation efficiency of a SBEIIa or SBEIIb gene are not expectedto alter the activity of the genes and are therefore regarded herein asnon-mutant, i.e. such genes are active variants and not encompassed in“mutant alleles”. Alternatively, the nucleotide substitution(s) maychange the encoded amino acid sequence and thereby alter the activity ofthe encoded enzyme, particularly if conserved amino acids aresubstituted for another amino acid which is quite different i.e. anon-conservative substitution. Typical conservative substitutions arethose made in accordance with Table 3.

The term “mutation” as used herein does not include silent nucleotidesubstitutions which do not affect the activity of the gene, andtherefore includes only alterations in the gene sequence which affectthe gene activity. The term “polymorphism” refers to any change in thenucleotide sequence including such silent nucleotide substitutions.Screening methods may first involve screening for polymorphisms andsecondly for mutations within a group of polymorphic variants.

As is understood in the art, hexaploid wheats such as bread wheatcomprise three genomes which are commonly designated the A, B and Dgenomes, while tetraploid wheats such as durum wheat comprise twogenomes commonly designated the A and B genomes. Each genome comprises 7pairs of chromosomes which may be observed by cytological methods duringmeiosis and thus identified, as is well known in the art.

The terms “plant(s)” and “wheat plant(s)” as used herein as a noungenerally refer to whole plants, but when “plant” or “wheat” is used asan adjective, the terms refer to any substance which is present in,obtained from, derived from, or related to a plant or a wheat plant,such as for example, plant organs (e.g. leaves, stems, roots, flowers),single cells (e.g. pollen), seeds, plant cells including for exampletissue cultured cells, products produced from the plant such as “wheatflour”, “wheat grain”, “wheat starch”, “wheat starch granules” and thelike. Plantlets and germinated seeds from which roots and shoots haveemerged are also included within the meaning of “plant”. The term “plantparts” as used herein refers to one or more plant tissues or organswhich are obtained from a whole plant, preferably a wheat plant. Plantparts include vegetative structures (for example, leaves, stems), roots,floral organs/structures, seed (including embryo, endosperm, and seedcoat), plant tissue (for example, vascular tissue, ground tissue, andthe like), cells and progeny of the same. The term “plant cell” as usedherein refers to a cell obtained from a plant or in a plant, preferablya wheat plant, and includes protoplasts or other cells derived fromplants, gamete-producing cells, and cells which regenerate into wholeplants. Plant cells may be cells in culture. By “plant tissue” is meantdifferentiated tissue in a plant or obtained from a plant (“explant”) orundifferentiated tissue derived from immature or mature embryos, seeds,roots, shoots, fruits, pollen, and various forms of aggregations ofplant cells in culture, such as calli. Plant tissues in or from seedssuch as wheat seeds are seed coat, endosperm, scutellum, aleurone layerand embryo.

Cereals as used herein means plants or grain of the monocotyledonousfamilies Poaceae or Graminae which are cultivated for the ediblecomponents of their seeds, and includes wheat, barley, maize, oats, rye,rice, sorghum, triticale, millet, buckwheat. Preferably, the cerealplant or grain is wheat or barley plant or grain, more preferably wheatplant or grain. In a further preferred embodiment, the cereal plant isnot rice or maize or both of these.

As used herein, the term “wheat” refers to any species of the GenusTriticum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. Wheat includes “hexaploid wheat”which has genome organization of AABBDD, comprised of 42 chromosomes,and “tetraploid wheat” which has genome organization of AABB, comprisedof 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, T.macha, T. compactum, T. sphaerococcum, T. vavilovii, and interspeciescross thereof. Tetraploid wheat includes T. durum (also referred to asdurum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T.dicoccum, T. polonicum, and interspecies cross thereof. In addition, theterm “wheat” includes possible progenitors of hexaploid or tetraploidTriticum sp. such as T. uartu, T. monococcum or T. boeoticum for the Agenome, Aegilops speltoides for the B genome, and T. tauschii (alsoknown as Aegilops squarrosa or Aegilops tauschii) for the D genome. Awheat cultivar for use in the present invention may belong to, but isnot limited to, any of the above-listed species. Also encompassed areplants that are produced by conventional techniques using Triticum sp.as a parent in a sexual cross with a non-Triticum species, such as ryeSecale cereale, including but not limited to Triticale. Preferably thewheat plant is suitable for commercial production of grain, such ascommercial varieties of hexaploid wheat or durum wheat, having suitableagronomic characteristics which are known to those skilled in the art.More preferably the wheat is Triticum aestivum ssp. aestivum or Triticumturgidum ssp. durum, and most preferably the wheat is Triticum aestivumssp. aestivum, herein also referred to as “breadwheat”.

As used herein, the term “barley” refers to any species of the GenusHordeum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Hordeum species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Hordeum vulgare or suitablefor commercial production of grain.

The wheat plants of the invention may have many uses other than uses forfood or animal feed, for example uses in research or breeding. In seedpropagated crops such as wheat, the plants can be self-crossed toproduce a plant which is homozygous for the desired genes, or haploidtissues such as developing germ cells can be induced to double thechromosome complement to produce a homozygous plant. The inbred wheatplant of the invention thereby produces seed containing the combinationof mutant SBEII alleles which may be homozygous. These seeds can begrown to produce plants that would have the selected phenotype such as,for example, high amylose content in its starch.

The wheat plants of the invention may be crossed with plants containinga more desirable genetic background, and therefore the inventionincludes the transfer of the low SBEII trait to other geneticbackgrounds. After the initial crossing, a suitable number ofbackcrosses may be carried out to remove a less desirable background.SBEII allele-specific PCR-based markers such as those described hereinmay be used to screen for or identify progeny plants or grain with thedesired combination of alleles, thereby tracking the presence of thealleles in the breeding program. The desired genetic background mayinclude a suitable combination of genes providing commercial yield andother characteristics such as agronomic performance or abiotic stressresistance. The genetic background might also include other alteredstarch biosynthesis or modification genes, for example genes from otherwheat lines. The genetic background may comprise one or more transgenessuch as, for example, a gene that confers tolerance to a herbicide suchas glyphosate.

The desired genetic background of the wheat plant will includeconsiderations of agronomic yield and other characteristics. Suchcharacteristics might include whether it is desired to have a winter orspring types, agronomic performance, disease resistance and abioticstress resistance. For Australian use, one might want to cross thealtered starch trait of the wheat plant of the invention into wheatcultivars such as Baxter, Kennedy, Janz, Frame, Rosella, Cadoux,Diamondbird or other commonly grown varieties. Other varieties will besuited for other growing regions. It is preferred that the wheat plantof the invention provide a grain yield of at least 80% relative to theyield of the corresponding wild-type variety in at least some growingregions, more preferably at least 85% or at least 90%, and even morepreferably at least 95% relative to a wild-type variety having about thesame genetic background, grown under the same conditions. Mostpreferably, the grain yield of the wheat plant of the invention is atleast as great as the yield of the wild-type wheat plant having aboutthe same genetic background, grown under the same conditions. The yieldcan readily be measured in controlled field trials, or in simulatedfield trials in the greenhouse, preferably in the field.

Marker assisted selection is a well recognised method of selecting forheterozygous plants obtained when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene(s) of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants.

Procedures such as crossing wheat plants, self-fertilising wheat plantsor marker-assisted selection are standard procedures and well known inthe art. Transferring alleles from tetraploid wheat such as durum wheatto a hexaploid, or other forms of hybridisation, is more difficult butis also known in the art.

To identify the desired phenotypic characteristic, wheat plants thatcontain a combination of mutant SBEIIa and SBEIIb alleles or otherdesired genes are typically compared to control plants. When evaluatinga phenotypic characteristic associated with enzyme activity such asamylose content in the grain starch, the plants to be tested and controlplants are grown under growth chamber, greenhouse, open top chamberand/or field conditions. Identification of a particular phenotypic traitand comparison to controls is based on routine statistical analysis andscoring. Statistical differences between plants lines can be assessed bycomparing—enzyme activity between plant lines within each tissue typeexpressing the enzyme. Expression and activity are compared to growth,development and yield parameters which include plant part morphology,colour, number, size, dimensions, dry and wet weight, ripening, above-and below-ground biomass ratios, and timing, rates and duration ofvarious stages of growth through senescence, including vegetativegrowth, fruiting, flowering, and soluble carbohydrate content includingsucrose, glucose, fructose and starch levels as well as endogenousstarch levels. Preferably, the wheat plants of the invention differ fromwild-type plants in one or more of these parameters by less than 50%,more preferably less than 40%, less than 30%, less than 20%, less than15%, less than 10%, less than 5%, less than 2% or less than 1% whengrown under the same conditions.

As used herein, the term “linked” refers to a marker locus and a secondlocus being sufficiently close on a chromosome that they will beinherited together in more than 50% of meioses, e.g., not randomly. Thisdefinition includes the situation where the marker locus and secondlocus form part of the same gene. Furthermore, this definition includesthe situation where the marker locus comprises a polymorphism that isresponsible for the trait of interest (in other words the marker locusis directly “linked” to the phenotype). The term “genetically linked” asused herein is narrower, only used in relation to where a marker locusand a second locus being sufficiently close on a chromosome that theywill be inherited together in more than 50% of meioses. Thus, thepercent of recombination observed between the loci per generation(centimorgans (cM)), will be less than 50. In particular embodiments ofthe invention, genetically linked loci may be 45, 35, 25, 15, 10, 5, 4,3, 2, or 1 or less cM apart on a chromosome. Preferably, the markers areless than 5 cM or 2 cM apart and most preferably about 0 cM apart. Asdescribed in Example 5 herein, the SBEIIa and SBEIIb genes aregenetically linked on the long arm of chromosome 2 of each of the wheatgenomes, being about 0.5 cM apart, which corresponds to about 100-200 kbin physical distance.

As used herein, the “other genetic markers” may be any molecules whichare linked to a desired trait in the wheat plants of the invention. Suchmarkers are well known to those skilled in the art and include molecularmarkers linked to genes determining traits such disease resistance,yield, plant morphology, grain quality, other dormancy traits such asgrain colour, gibberellic acid content in the seed, plant height, flourcolour and the like. Examples of such genes are stem-rust resistancegenes Sr2 or Sr38, the stripe rust resistance genes Yr10 or Yr17, thenematode resistance genes such as Cre1 and Cre3, alleles at gluteninloci that determine dough strength such as Ax, Bx, Dx, Ay, By and Dyalleles, the Rht genes that determine a semi-dwarf growth habit andtherefore lodging resistance (Eagles et al., 2001; Langridge et al.,2001; Sharp et al., 2001).

The wheat plants, wheat plant parts and products therefrom of theinvention are preferably non-transgenic for genes that inhibitexpression of SBEIIa i.e. they do not comprise a transgene encoding anRNA molecule that reduces expression of the endogenous SBEIIa genes,although in this embodiment they may comprise other transgenes, eg.herbicide tolerance genes. More preferably, the wheat plant, grain andproducts therefrom are non-transgenic, i.e. they do not contain anytransgene, which is preferred in some markets. Such products are alsodescribed herein as “non-transformed” products. Such non-transgenicplants and grain comprise the multiple mutant SBEII alleles as describedherein, such as those produced after mutagenesis.

The terms “transgenic plant” and “transgenic wheat plant” as used hereinrefer to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar. That is,transgenic plants (transformed plants) contain genetic material thatthey did not contain prior to the transformation. A “transgene” asreferred to herein has the normal meaning in the art of biotechnologyand refers to a genetic sequence which has been produced or altered byrecombinant DNA or RNA technology and which has been introduced into theplant cell. The transgene may include genetic sequences obtained from orderived from a plant cell, or another plant cell, or a non-plant source,or a synthetic sequence. Typically, the transgene has been introducedinto the plant by human manipulation such as, for example, bytransformation but any method can be used as one of skill in the artrecognizes. The genetic material is typically stably integrated into thegenome of the plant. The introduced genetic material may comprisesequences that naturally occur in the same species but in a rearrangedorder or in a different arrangement of elements, for example anantisense sequence. Plants containing such sequences are included hereinin “transgenic plants”. Transgenic plants as defined herein include allprogeny of an initial transformed and regenerated plant (T0 plant) whichhas been genetically modified using recombinant techniques, where theprogeny comprise the transgene. Such progeny may be obtained byself-fertilisation of the primary transgenic plant or by crossing suchplants with another plant of the same species. In an embodiment, thetransgenic plants are homozygous for each and every gene that has beenintroduced (transgene) so that their progeny do not segregate for thedesired phenotype. Transgenic plant parts include all parts and cells ofsaid plants which comprise the transgene such as, for example, seeds,cultured tissues, callus and protoplasts. A “non-transgenic plant”,preferably a non-transgenic wheat plant, is one which has not beengenetically modified by the introduction of genetic material byrecombinant DNA techniques.

As used herein, the term “corresponding non-transgenic plant” refers toa plant which is the same or similar in most characteristics, preferablyisogenic or near-isogenic relative to the transgenic plant, but withoutthe transgene of interest. Preferably, the corresponding non-transgenicplant is of the same cultivar or variety as the progenitor of thetransgenic plant of interest, or a sibling plant line which lacks theconstruct, often termed a “segregant”, or a plant of the same cultivaror variety transformed with an “empty vector” construct, and may be anon-transgenic plant. “Wild-type”, as used herein, refers to a cell,tissue or plant that has not been modified according to the invention.Wild-type cells, tissue or plants known in the art and may be used ascontrols to compare levels of expression of an exogenous nucleic acid orthe extent and nature of trait modification with cells, tissue or plantsmodified as described herein. As used herein, “wild-type wheat grain”means a corresponding non-mutagenized, non-transgenic wheat grain.Specific wild-type wheat grains as used herein include but are notlimited to Sunstate and Cadoux.

Any of several methods may be employed to determine the presence of atransgene in a transformed plant. For example, polymerase chain reaction(PCR) may be used to amplify sequences that are unique to thetransformed plant, with detection of the amplified products by gelelectrophoresis or other methods. DNA may be extracted from the plantsusing conventional methods and the PCR reaction carried out usingprimers that will distinguish the transformed and non-transformedplants. An alternative method to confirm a positive transformant is bySouthern blot hybridization, well known in the art. Wheat plants whichare transformed may also be identified i.e. distinguished fromnon-transformed or wild-type wheat plants by their phenotype, forexample conferred by the presence of a selectable marker gene, or byimmunoassays that detect or quantify the expression of an enzyme encodedby the transgene, or any other phenotype conferred by the transgene.

The wheat plants of the present invention may be grown or harvested forgrain, primarily for use as food for human consumption or as animalfeed, or for fermentation or industrial feedstock production such asethanol production, among other uses. Alternatively, the wheat plantsmay be used directly as feed. The plant of the present invention ispreferably useful for food production and in particular for commercialfood production. Such food production might include the making of flour,dough, semolina or other products from the grain that might be aningredient in commercial food production.

As used herein, the term “grain” generally refers to mature, harvestedseed of a plant but can also refer to grain after imbibition orgermination, according to the context. Mature cereal grain such as wheatcommonly has a moisture content of less than about 18-20%. As usedherein, the term “seed” includes harvested seed but also includes seedwhich is developing in the plant post anthesis and mature seed comprisedin the plant prior to harvest.

As used herein, “germination” refers to the emergence of the root tipfrom the seed coat after imbibition. “Germination rate” refers to thepercentage of seeds in a population which have germinated over a periodof time, for example 7 or 10 days, after imbibition. Germination ratescan be calculated using techniques known in the art. For example, apopulation of seeds can be assessed daily over several days to determinethe germination percentage over time. With regard to grain of thepresent invention, as used herein the term “germination rate which issubstantially the same” means that the germination rate of the grain isat least 90%, that of corresponding wild-type grain.

Starch is readily isolated from wheat grain using standard methods, forexample the method of Schulman and Kammiovirta, 1991. On an industrialscale, wet or dry milling can be used. Starch granule size is importantin the starch processing industry where there is separation of thelarger A granules from the smaller B granules.

Wild-type wheat grown commercially has a starch content in the grainwhich is usually in the range 55-65%, depending somewhat on the cultivargrown. In comparison, the seed or grain of the invention has a starchcontent of at least 90% relative to that of wild-type grain, andpreferably at least 93%, at least 95%, or at least 98% relative to thestarch content of wild-type grain when the plants are grown under thesame conditions. In further embodiments, the starch content of the grainis at least about 25%, at least about 35%, at least about 45%, or atleast about 55% to about 65% as a percentage of the grain weight (w/w).Other desirable characteristics include the capacity to mill the grain,in particular the grain hardness. Another aspect that might make a wheatplant of higher value is the degree of starch extraction from the grain,the higher extraction rates being more useful. Grain shape is alsoanother feature that can impact on the commercial usefulness of a plant,thus grain shape can have an impact on the ease or otherwise with whichthe grain can be milled.

In another aspect, the invention provides starch granules or starchobtained from the grain of the plant as described above, having anincreased proportion of amylose and a reduced proportion of amylopectin.Purified starch may be obtained from grain by a milling process, forexample a wet milling process, which involves the separation of thestarch from protein, oil and fibre. The initial product of the millingprocess is a mixture or composition of starch granules, and theinvention therefore encompasses such granules. The starch granules fromwheat comprise starch granule-bound proteins including GBSS, SBEIIa andSBEIIb amongst other proteins and therefore the presence of theseproteins distinguish wheat starch granules from starch granules of othercereals. The starch from starch granules may be purified by removal ofthe proteins after disruption and dispersal of the starch granules byheat and/or chemical treatment. The starch granules from the wheat grainof the invention are typically distorted in shape and surfacemorphology, when observed under light microscopy, as exemplified herein,particularly for wheat grain having an amylose content of at least 50%as a percentage of the total starch of the grain. In an embodiment, atleast 50%, preferably at least 60% or at least 70% of the starchgranules obtained from the grain show distorted shape or surfacemorphology. The starch granules also show a loss of birefringence whenobserved under polarised light.

The starch of the grain, the starch of the starch granules, and thepurified starch of the invention may be further characterized by one ormore of the following properties:

-   -   (i) at least 50% (w/w), or at least 60% (w/w), or at least 67%        (w/w) amylose as a proportion of the total starch;    -   (ii) modified swelling volume;    -   (iii) modified chain length distribution and/or branching        frequency;    -   (iv) modified gelatinisation temperature;    -   (v) modified viscosity (peak viscosity, pasting temperature,        etc.);    -   (vi) modified molecular mass of amylopectin and/or amylose;    -   (vii) modified % crystallinity    -   (viii) comprising at least 2% resistant starch; and/or    -   (ix) comprising a low relative glycaemic index (GI).

The starch may also be characterized by its swelling volume in heatedexcess water compared to wild-type starch. Swelling volume is typicallymeasured by mixing either a starch or flour with excess water andheating to elevated temperatures, typically greater than 90° C. Thesample is then collected by centrifugation and the swelling volume isexpressed as the mass of the sedimented material divided by the dryweight of the sample. A low swelling characteristic is useful where itis desired to increase the starch content of a food preparation, inparticular a hydrated food preparation.

One measure of an altered amylopectin structure is the distribution ofchain lengths, or the degree of polymerization, of the starch. The chainlength distribution may be determined by using fluorophore-assistedcarbohydrate electrophoresis (FACE) following isoamylose de-branching.The amylopectin of the starch of the invention may have a distributionof chain length in the range from 5 to 60 that is greater than thedistribution of starch from wild-type plants upon debranching. Starchwith longer chain lengths will also have a commensurate decrease infrequency of branching. Thus the starch may also have a distribution oflonger amylopectin chain lengths in the amylopectin still present. Theamylopectin of the grain may be characterised in comprising a reducedproportion of the 4-12 dp chain length fraction relative to theamylopectin of wild-type grain, as measured after isoamylase debranchingof the amylopectin.

In another aspect of the invention, the wheat starch may have an alteredgelatinisation temperature, which may be readily measured bydifferential scanning calorimetry (DSC). Gelatinisation is theheat-driven collapse (disruption) of molecular order within the starchgranule in excess water, with concomitant and irreversible changes inproperties such as granular swelling, crystallite melting, loss ofbirefringence, viscosity development and starch solubilisation. Thegelatinisation temperature may be either increased or decreased comparedto starch from wild-type plants, depending on the chain length of theremaining amylopectin. High amylose starch from amylose extender (ae)mutants of maize showed a higher gelatinisation temperature than normalmaize (Fuwa et al., 1999; Krueger et al., 1987). On the other hand,starch from barley sex6 mutants that lack starch synthase IIa activityhad lower gelatinisation temperatures and the enthalpy for thegelatinisation peak was reduced when compared to that from controlplants (Morell et al., 2003).

The gelatinisation temperature, in particular the temperature of onsetof the first peak or the temperature for the apex of the first peak, maybe elevated by at least 3° C., preferably at least 5° C. or morepreferably at least 7° C. as measured by DSC compared to starchextracted from a similar, but unaltered grain. The starch may comprisean elevated level of resistant starch, with an altered structureindicated by specific physical characteristics including one or more ofthe group consisting of physical inaccessibility to digestive enzymeswhich may be by reason of having altered starch granule morphology, thepresence of appreciable starch associated lipid, altered crystallinity,and altered amylopectin chain length distribution. The high proportionof amylose also contributes to the level of resistant starch.

The starch structure of the wheat of the present invention may alsodiffer in that the degree of crystallinity is reduced compared to normalstarch isolated from wheat. The reduced crystallinity of a starch isalso thought to be associated with enhance organoleptic properties andcontributes to a smoother mouth feel. Thus, the starch may additionallyexhibit reduced crystallinity resulting from reduced levels of activityof one or more amylopectin synthesis enzymes. Crystallinity is typicallyinvestigated by X-ray crystallography.

In some embodiments, the present starch provides modified digestiveproperties such as increased resistant starch including between 1% to20%, 2% to 18%, 3% to 18% or 5% to 15% resistant starch and a decreasedGlycaemic Index (GI).

The invention also provides flour, meal or other products produced fromthe grain. These may be unprocessed or processed, for example byfractionation or bleaching.

The invention also provides starch from grain of the exemplified wheatplants comprising increased amounts of dietary fibre, preferably incombination with an elevated level of resistant starch. This increase isalso at least in part a result of the high relative level of amylose.

The term “dietary fibre” as used herein includes the carbohydrate andcarbohydrate digestion products which are not absorbed in the smallintestine of healthy humans but which enter the large bowel. Thisincludes resistant starch and other soluble and insoluble carbohydratepolymers. It is intended to comprise that portion of carbohydrates thatare fermentable, at least partially, in the large bowel by the residentmicroflora. The starch of the invention contains relatively high levelsof dietary fibre, more particularly amylose. The dietary fibre contentof the grain of the present invention results at least in part from theincreased amylose content in the starch of the grain, and also, or incombination with an increased resistant starch content as a percentageof the total starch. “Resistant starch” is defined herein as the sum ofstarch and products of starch digestion not absorbed in the smallintestine of healthy humans but entering into the large bowel. This isdefined in terms of a percentage of the total starch of the grain, or apercentage of the total starch content in the food, according to thecontext. Thus, resistant starch excludes products digested and absorbedin the small intestine. Resistant starches include physicallyinaccessible starch (RS1 form), resistant native starch granules (RS2),retrograded starches (RS3), and chemically modified starches (RS4). Thealtered starch structure and in particular the high amylose levels ofthe starch of the invention give rise to an increase in resistant starchwhen consumed in food. The starch may be in an RS1 form, being somewhatinaccessible to digestion. Starch-lipid association as measured byV-complex crystallinity is also likely to contribute to the level ofresistant starch.

Whilst the invention may be particularly useful in the treatment orprophylaxis of humans, it is to be understood that the invention is alsoapplicable to non-human subjects including but not limited toagricultural animals such as cows, sheep, pigs and the like, domesticanimals such as dogs or cats, laboratory animals such as rabbits orrodents such as mice, rats, hamsters, or animals that might be used forsport such as horses. The method may be particularly applicable tonon-ruminant mammals or animals such as mono-gastric mammals. Theinvention may also be applicable to other agricultural animals forexample poultry including, for example, chicken, geese, ducks, turkeys,or quails, or fish.

The method of treating the subject, particularly humans, may comprisethe step of administering altered wheat grain, flour, starch or a foodor drink product as defined herein to the subject, in one or more doses,in an amount and for a period of time whereby the level of the one ormore of the bowel health or metabolic indicators improves. The indicatormay change relative to consumption of non-altered wheat starch or wheator product thereof, within a time period of hours, as in the case ofsome of the indicators such as pH, elevation of levels of SCFA,post-prandial glucose fluctuation, or it may take days such as in thecase of increase in fecal bulk or improved laxation, or perhaps longerin the order of weeks or months such as in the case where the butyrateenhanced proliferation of normal colonocytes is measured. It may bedesirable that administration of the altered starch or wheat or wheatproduct be lifelong. However, there are good prospects for compliance bythe individual being treated given the relative ease with which thealtered starch can be administered.

Dosages may vary depending on the condition being treated or preventedbut are envisaged for humans as being at least 1 g of wheat grain orstarch of the invention per day, more preferably at least 2 g per day,preferably at least 10 or at least 20 g per day. Administration ofgreater than about 100 grams per day may require considerable volumes ofdelivery and reduce compliance. Most preferably the dosage for a humanis between 5 and 60 g of wheat grain or starch per day, or for adultsbetween 5 and 100 g per day.

Glycaemic Index (GI) relates to the rate of digestion of foodscomprising the starch, and is a comparison of the effect of a test foodwith the effect of white bread or glucose on excursions in blood glucoseconcentration. The Glycaemic Index is a measure of the likely effect ofthe food concerned on post prandial serum glucose concentration anddemand for insulin for blood glucose homeostasis. One importantcharacteristic provided by foods of the invention is a reduced glycaemicindex. Serum glucose levels were lower 30 min after ingestion of highamylose wheat products by human volunteers compared to low amylose wheat(Goddard et al., 1984). Furthermore, the foods may have a low level offinal digestion and consequently be relatively low-calorie. A lowcalorific product might be based on inclusion of flour produced frommilled wheat grain. Such foods may have the effect of being filling,enhancing bowel health, reducing the post-prandial serum glucose andlipid concentration as well as providing for a low calorific foodproduct.

The indicators of improved bowel health may comprise, but are notnecessarily limited to:

-   -   i) decreased pH of the bowel contents,    -   ii) increased total SCFA concentration or total SCFA amount in        the bowel contents,    -   iii) increased concentration or amount of one or more SCFAs in        the bowel contents,    -   iv) increased fecal bulk,    -   v) increase in total water volume of bowel or faeces, without        diarrhea,    -   vi) improved laxation,    -   vii) increase in number or activity of one or more species of        probiotic bacteria,    -   viii) increase in fecal bile acid excretion,    -   ix) reduced urinary levels of putrefactive products,    -   x) reduced fecal levels of putrefactive products,    -   xi) increased proliferation of normal colonocytes,    -   xii) reduced inflammation in the bowel of individuals with        inflamed bowel,    -   xiii) reduced fecal or large bowel levels of any one of urea,        creatinine and phosphate in uremic patients, and    -   xiv) any combination of the above.        The indicators of improved metabolic health may comprise, but        are not necessarily limited to:    -   i) stabilisation of post-prandial glucose fluctuation,    -   ii) improved (lowered) glycaemic response,    -   iii) reduced pro-prandial plasma insulin concentration,    -   iv) improved blood lipid profile,    -   v) lowering of plasma LDL cholesterol,    -   vi) reduced plasma levels of one or more of urea, creatinine and        phosphate in uremic patients,    -   vii) an improvement in a dysglucaemic response, or    -   viii) any combination of the above.

It will be understood that one benefit of the present invention is thatit provides for products such as bread that are of particularnutritional benefit, and moreover it does so without the need topost-harvest modify the starch or other constituents of the wheat grain.However, it may be desired to make modifications to the starch or otherconstituent of the grain, and the invention encompasses such a modifiedconstituent. Methods of modification are well known and include theextraction of the starch or other constituent by conventional methodsand modification of the starches to increase the resistant form. Thestarch may be modified by treatment with heat and/or moisture,physically (for example ball milling), enzymatically (using for exampleα- or β-amylase, pullalanase or the like), chemical hydrolysis (wet ordry using liquid or gaseous reagents), oxidation, cross bonding withdifunctional reagents (for example sodium trimetaphosphate, phosphorusoxychloride), or carboxymethylation.

The wheat starch of the present invention will be a suitable substratefor fermentation for ethanol (biofuel) or ethanol-containing beveragesand the wheat grain or wheat starch for other fermentation products suchas foods, nutraceuticals (insoluble or soluble fibre), enzymes andindustrial materials. The methods for fermentation using plant-derivedstarch are well known to those skilled in the art, with establishedprocesses for various fermentation products (see for example Vogel etal., 1996 and references cited therein). In one embodiment, the starchcarbohydrates may be extracted by crushing the wheat plant parts of theinvention such as grain, or by diffusion from the plant tissues intowater or another suitable solvent. Wheat tissues or starch of theinvention may be used directly as a substrate for fermentation orbioconversion in a batch, continuous, or immobilized-cell process.

The terms “polypeptide” and “protein” are generally used interchangeablyherein. The terms “proteins” and “polypeptides” as used herein alsoinclude variants, mutants, modifications and/or derivatives of thepolypeptides of the invention as described herein. As used herein,“substantially purified polypeptide” refers to a polypeptide that hasbeen separated from the lipids, nucleic acids, other peptides and othermolecules with which it is associated in its native state. Preferably,the substantially purified polypeptide is at least 60% free, morepreferably at least 75% free, and more preferably at least 90% free fromother components with which it is naturally associated. By “recombinantpolypeptide” is meant a polypeptide made using recombinant techniques,i.e., through the expression of a recombinant polynucleotide in a cell,preferably a plant cell and more preferably a wheat cell. In anembodiment, the polypeptide has starch branching enzyme activity,particularly SBEII activity, and is at least 90% identical to a SBEIIdescribed herein.

As used herein a “biologically active” fragment is a portion of apolypeptide of the invention which maintains a defined activity of thefull-length polypeptide. In a particularly preferred embodiment, thebiologically active fragment has starch branching enzyme activity.Biologically active fragments can be any size as long as they maintainthe defined activity, but are preferably at least 700 or 800 amino acidresidues long. The % identity of a polypeptide relative to anotherpolypeptide can be determined by GAP (Needleman and Wunsch, 1970)analysis (GCG program) with a gap creation penalty=5, and a gapextension penalty=0.3. The query sequence is at least 50 amino acids inlength, and the GAP analysis aligns the two sequences over a region ofat least 50 amino acids. More preferably, the query sequence is at least100 amino acids in length and the GAP analysis aligns the two sequencesover a region of at least 100 amino acids. Even more preferably, thequery sequence is at least 250 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 250 aminoacids. Most preferably, two SBEII polypeptides are aligned over theirfull length amino acid sequences.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 75%, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, more preferablyat least 93%, more preferably at least 94%, more preferably at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, more preferably at least 99%, more preferablyat least 99.1%, more preferably at least 99.2%, more preferably at least99.3%, more preferably at least 99.4%, more preferably at least 99.5%,more preferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention or by mutagenesis in vivo such asby chemical or radiation treatment. Such mutants include, for example,deletions, insertions or substitutions of residues within the amino acidsequence. The polynucleotides of the invention may be subjected to DNAshuffling techniques as described by Harayama, 1998 or other in vitromethods to produce altered polynucleotides which encode polypeptidevariants. These DNA shuffling techniques may use genetic sequencesrelated to those of the present invention, such as SBE genes from plantspecies other than wheat. Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess, for example, SBE activity.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active site(s). Other sites of interestare those in which particular residues obtained from various strains orspecies are identical i.e. conserved amino acids. These positions may beimportant for biological activity. These amino acids, especially thosefalling within a contiguous sequence of at least three other identicallyconserved amino acids, are preferably substituted in a relativelyconservative manner in order to retain function such as SBEII activity.Such conservative substitutions are shown in Table 1 under the headingof “exemplary substitutions”. “Non-conservative amino acidsubstitutions” are defined herein as substitutions other than thoselisted in Table 3 (Exemplary conservative substitutions).Non-conservative substitutions in an SBEII are expected to reduce theactivity of the enzyme and many will correspond to an SBEII encoded by a“partial loss of function mutant SBEII gene”.

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by phosphorylation, as has been shown for SBEI, SBEIIaand SBEIIb in amyloplasts of wheat (Tetlow et al., 2004). Thesemodifications may serve to regulate the activity of the enzyme, forexample by regulating the formation of protein complexes in amyloplastsduring starch synthesis (Tetlow et al., 2008), or to increase thestability and/or bioactivity of the polypeptide of the invention, orserve as a ligand for binding of another molecule.

In some embodiments, the present invention involves modification of geneactivity, particularly of SBEII gene activity, combinations of mutantgenes, and the construction and use of chimeric genes. As used herein,the term “gene” includes any deoxyribonucleotide sequence which includesa protein coding region or which is transcribed in a cell but nottranslated, together with associated non-coding and regulatory regions.Such associated regions are typically located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of about 2 kb on eitherside. In this regard, the gene includes control signals such aspromoters, enhancers, transcription termination and/or polyadenylationsignals that are naturally associated with a given gene, or heterologouscontrol signals in which case the gene is referred to as a “chimericgene”. The sequences which are located 5′ of the protein coding regionand which are present on the mRNA are referred to as 5′ non-translatedsequences. The sequences which are located 3′ or downstream of theprotein coding region and which are present on the mRNA are referred toas 3′ non-translated sequences. The term “gene” encompasses both cDNAand genomic forms of a gene. The term “gene” includes synthetic orfusion molecules encoding the proteins of the invention describedherein. Genes are ordinarily present in the wheat genome asdouble-stranded DNA. A chimeric gene may be introduced into anappropriate vector for extrachromosomal maintenance in a cell or forintegration into the host genome. Genes or genotypes as referred toherein in italicised form (e.g. SBEIIa) while proteins, enzymes orphenotypes are referred to in non-italicised form (SBEIIa).

A genomic form or clone of a gene containing the coding region may beinterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” An “intron” as used herein is asegment of a gene which is transcribed as part of a primary RNAtranscript but is not present in the mature mRNA molecule. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA). Introns may containregulatory elements such as enhancers. “Exons” as used herein refer tothe DNA regions corresponding to the RNA sequences which are present inthe mature mRNA or the mature RNA molecule in cases where the RNAmolecule is not translated. An mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.

The present invention refers to various polynucleotides. As used herein,a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means apolymer of nucleotides, which may be DNA or RNA or a combinationthereof, for example a heteroduplex of DNA and RNA, and includes forexample mRNA, cRNA, cDNA, tRNA, siRNA, shRNA, hpRNA, and single ordouble-stranded DNA. It may be DNA or RNA of cellular, genomic orsynthetic origin, for example made on an automated synthesizer, and maybe combined with carbohydrate, lipids, protein or other materials,labelled with fluorescent or other groups, or attached to a solidsupport to perform a particular activity defined herein. Preferably thepolynucleotide is solely DNA or solely RNA as occurs in a cell, and somebases may be methylated or otherwise modified as occurs in a wheat cell.The polymer may be single-stranded, essentially double-stranded orpartly double-stranded. An example of a partly-double stranded RNAmolecule is a hairpin RNA (hpRNA), short hairpin RNA (shRNA) orself-complementary RNA which include a double stranded stem formed bybasepairing between a nucleotide sequence and its complement and a loopsequence which covalently joins the nucleotide sequence and itscomplement. Basepairing as used herein refers to standard basepairingbetween nucleotides, including G:U basepairs in an RNA molecule.“Complementary” means two polynucleotides are capable of basepairingalong part of their lengths, or along the full length of one or both.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Asused herein, an “isolated polynucleotide” or “isolated nucleic acidmolecule” means a polynucleotide which is at least partially separatedfrom, preferably substantially or essentially free of, thepolynucleotide sequences of the same type with which it is associated orlinked in its native state. For example, an “isolated polynucleotide”includes a polynucleotide which has been purified or separated from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment. Preferably, the isolated polynucleotide isalso at least 90% free from other components such as proteins,carbohydrates, lipids etc. The term “recombinant polynucleotide” as usedherein refers to a polynucleotide formed in vitro by the manipulation ofnucleic acid into a form not normally found in nature. For example, therecombinant polynucleotide may be in the form of an expression vector.Generally, such expression vectors include transcriptional andtranslational regulatory nucleic acid operably connected to thenucleotide sequence to be transcribed in the cell.

The present invention refers to use of oligonucleotides which may beused as “probes” or “primers”. As used herein, “oligonucleotides” arepolynucleotides up to 50 nucleotides in length. They can be RNA, DNA, orcombinations or derivatives of either. Oligonucleotides are typicallyrelatively short single stranded molecules of 10 to 30 nucleotides,commonly 15-25 nucleotides in length, typically comprised of 10-30 or15-25 nucleotides which are identical to, or complementary to, part ofan SBEIIa or SBEIIb gene or cDNA corresponding to an SBEIIa or SBEIIbgene. When used as a probe or as a primer in an amplification reaction,the minimum size of such an oligonucleotide is the size required for theformation of a stable hybrid between the oligonucleotide and acomplementary sequence on a target nucleic acid molecule. Preferably,the oligonucleotides are at least 15 nucleotides, more preferably atleast 18 nucleotides, more preferably at least 19 nucleotides, morepreferably at least 20 nucleotides, even more preferably at least 25nucleotides in length. Polynucleotides used as a probe are typicallyconjugated with a detectable label such as a radioisotope, an enzyme,biotin, a fluorescent molecule or a chemiluminescent molecule.Oligonucleotides and probes of the invention are useful in methods ofdetecting an allele of a SBEIIa, SBEIIb or other gene associated with atrait of interest, for example modified starch. Such methods employnucleic acid hybridization and in many instances include oligonucleotideprimer extension by a suitable polymerase, for example as used in PCRfor detection or identification of wild-type or mutant alleles.Preferred oligonucleotides and probes hybridise to a SBEIIa or SBEIIbgene sequence from wheat, including any of the sequences disclosedherein, for example SEQ ID NOs: 36 to 149. Preferred oligonucleotidepairs are those that span one or more introns, or a part of an intronand therefore may be used to amplify an intron sequence in a PCRreaction. Numerous examples are provided in the Examples herein.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence and which are able to function in ananalogous manner to, or with the same activity as, the referencesequence. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide, or that have, when comparedto naturally occurring molecules, one or more mutations. Accordingly,the terms “polynucleotide variant” and “variant” include polynucleotidesin which one or more nucleotides have been added or deleted, or replacedwith different nucleotides. In this regard, it is well understood in theart that certain alterations inclusive of mutations, additions,deletions and substitutions can be made to a reference polynucleotidewhereby the altered polynucleotide retains the biological function oractivity of the reference polynucleotide. Accordingly, these termsencompass polynucleotides that encode polypeptides that exhibitenzymatic or other regulatory activity, or polynucleotides capable ofserving as selective probes or other hybridising agents. The terms“polynucleotide variant” and “variant” also include naturally occurringallelic variants. Mutants can be either naturally occurring (that is tosay, isolated from a natural source) or synthetic (for example, byperforming site-directed mutagenesis on the nucleic acid). Preferably, apolynucleotide variant of the invention which encodes a polypeptide withenzyme activity is greater than 400, more preferably greater than 500,more preferably greater than 600, more preferably greater than 700, morepreferably greater than 800, more preferably greater than 900, and evenmore preferably greater than 1,000 nucleotides in length, up to the fulllength of the gene.

A variant of an oligonucleotide of the invention includes molecules ofvarying sizes which are capable of hybridising, for example, to thewheat genome at a position close to that of the specific oligonucleotidemolecules defined herein. For example, variants may comprise additionalnucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as longas they still hybridise to the target region. Furthermore, a fewnucleotides may be substituted without influencing the ability of theoligonucleotide to hybridise to the target region. In addition, variantsmay readily be designed which hybridise close (for example, but notlimited to, within 50 nucleotides) to the region of the plant genomewhere the specific oligonucleotides defined herein hybridise.

By “corresponds to” or “corresponding to” in the context ofpolynucleotides or polypeptides is meant a polynucleotide (a) having anucleotide sequence that is substantially identical or complementary toall or a portion of a reference polynucleotide sequence or (b) encodingan amino acid sequence identical to an amino acid sequence in a peptideor protein. This phrase also includes within its scope a peptide orpolypeptide having an amino acid sequence that is substantiallyidentical to a sequence of amino acids in a reference peptide orprotein. Terms used to describe sequence relationships between two ormore polynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity”, “substantial identity” and “identical”, and are defined withrespect to a defined minimum number of nucleotides or amino acidresidues or preferably over the full length. The terms “sequenceidentity” and “identity” are used interchangeably herein to refer to theextent that sequences are identical on a nucleotide-by-nucleotide basisor an amino acid-by-amino acid basis over a window of comparison. Thus,a “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

The % identity of a polynucleotide can be determined by GAP (Needlemanand Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5,and a gap extension penalty=0.3. Unless stated otherwise, the querysequence is at least 45 nucleotides in length, and the GAP analysisaligns the two sequences over a region of at least 45 nucleotides.Preferably, the query sequence is at least 150 nucleotides in length,and the GAP analysis aligns the two sequences over a region of at least150 nucleotides. More preferably, the query sequence is at least 300nucleotides in length and the GAP analysis aligns the two sequences overa region of at least 300 nucleotides, or at least 400, 500 or 600nucleotides in each case. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997. Adetailed discussion of sequence analysis can be found in Unit 19.3 ofAusubel et al., 1994-1998, Chapter 15.

Nucleotide or amino acid sequences are indicated as “essentiallysimilar” when such sequences have a sequence identity of at least about95%, particularly at least about 98%, more particularly at least about98.5%, quite particularly about 99%, especially about 99.5%, moreespecially about 100%, quite especially are identical. It is clear thatwhen RNA sequences are described as essentially similar to, or have acertain degree of sequence identity with, DNA sequences, thymine (T) inthe DNA sequence is considered equal to uracil (U) in the RNA sequence.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 75%, more preferably at least80%, more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, more preferablyat least 93%, more preferably at least 94%, more preferably at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, more preferably at least 99%, more preferablyat least 99.1%, more preferably at least 99.2%, more preferably at least99.3%, more preferably at least 99.4%, more preferably at least 99.5%,more preferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

In some embodiments, the present invention refers to the stringency ofhybridization conditions to define the extent of complementarity of twopolynucleotides. “Stringency” as used herein, refers to the temperatureand ionic strength conditions, and presence or absence of certainorganic solvents, during hybridization. The higher the stringency, thehigher will be the degree of complementarity between a target nucleotidesequence and the labelled polynucleotide sequence. “Stringentconditions” refers to temperature and ionic conditions under which onlynucleotide sequences having a high frequency of complementary bases willhybridize. As used herein, the term “hybridizes under low stringency,medium stringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, hereinincorporated by reference. Specific hybridization conditions referred toherein are as follows: 1) low stringency hybridization conditions in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by twowashes in 0.2×SSC, 0.1% SDS at 50-55° C.; 2) medium stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringencyhybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

As used herein, a “chimeric gene” or “genetic construct” refers to anygene that is not a native gene in its native location i.e. it has beenartificially manipulated, including a chimeric gene or genetic constructwhich is integrated into the wheat genome. Typically a chimeric gene orgenetic construct comprises regulatory and transcribed or protein codingsequences that are not found together in nature. Accordingly, a chimericgene or genetic construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. The term“endogenous” is used herein to refer to a substance that is normallyproduced in an unmodified plant at the same developmental stage as theplant under investigation, preferably a wheat plant, such as starch or aSBEIIa or SBEIIb. An “endogenous gene” refers to a native gene in itsnatural location in the genome of an organism, preferably a SBEIIa orSBEIIb gene in a wheat plant. As used herein, “recombinant nucleic acidmolecule” refers to a nucleic acid molecule which has been constructedor modified by recombinant DNA technology. The terms “foreignpolynucleotide” or “exogenous polynucleotide” or “heterologouspolynucleotide” and the like refer to any nucleic acid which isintroduced into the genome of a cell by experimental manipulations,preferably the wheat genome, but which does not naturally occur in thecell. These include modified forms of gene sequences found in that cellso long as the introduced gene contains some modification, e.g. anintroduced mutation or the presence of a selectable marker gene,relative to the naturally-occurring gene. Foreign or exogenous genes maybe genes found in nature that are inserted into a non-native organism,native genes introduced into a new location within the native host, orchimeric genes or genetic constructs. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The term“genetically modified” includes introducing genes into cells, mutatinggenes in cells and altering or modulating the regulation of a gene in acell or organisms to which these acts have been done or their progeny.

The present invention refers to elements which are operably connected orlinked. “Operably connected” or “operably linked” and the like refer toa linkage of polynucleotide elements in a functional relationship.Typically, operably connected nucleic acid sequences are contiguouslylinked and, where necessary to join two protein coding regions,contiguous and in reading frame. A coding sequence is “operablyconnected to” another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single RNA, which iftranslated is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein, the term “cis-acting sequence”, “cis-acting element” or“cis-regulatory region” or “regulatory region” or similar term shall betaken to mean any sequence of nucleotides which regulates the expressionof the genetic sequence. This may be a naturally occurring cis-actingsequence in its native context, for example regulating a wheat SBEIIa orSBEIIb gene, or a sequence in a genetic construct which when positionedappropriately relative to an expressible genetic sequence, regulates itsexpression. Such a cis-regulatory region may be capable of activating,silencing, enhancing, repressing or otherwise altering the level ofexpression and/or cell-type-specificity and/or developmental specificityof a gene sequence at the transcriptional or post-transcriptional level.In preferred embodiments of the present invention, the cis-actingsequence is an activator sequence that enhances or stimulates theexpression of an expressible genetic sequence, such as a promoter. Thepresence of an intron in the 5′-leader (UTR) of genes has been shown toenhance expression of genes in monocotyledonous plants such as wheat(Tanaka et al., 1990). Another type of cis-acting sequence is a matrixattachment region (MAR) which may influence gene expression by anchoringactive chromatin domains to the nuclear matrix.

“Operably connecting” a promoter or enhancer element to a transcribablepolynucleotide means placing the transcribable polynucleotide (e.g.,protein-encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionof that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide, which isapproximately the same as the distance between that promoter and thegene it controls in its natural setting; i.e., the gene from which thepromoter is derived. As is known in the art, some variation in thisdistance can be accommodated without loss of function.

The present invention makes use of vectors for production, manipulationor transfer of chimeric genes or genetic constructs. By “vector” ismeant a nucleic acid molecule, preferably a DNA molecule derived, forexample, from a plasmid, bacteriophage or plant virus, into which anucleic acid sequence may be inserted. A vector preferably contains oneor more unique restriction sites and may be capable of autonomousreplication in a defined host cell including a target cell or tissue ora progenitor cell or tissue thereof, or be integrable into the genome ofthe defined host such that the cloned sequence is reproducible.Accordingly, the vector may be an autonomously replicating vector, i.e.,a vector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a linear orclosed circular plasmid, an extrachromosomal element, a minichromosome,or an artificial chromosome. The vector may contain any means forassuring self-replication. Alternatively, the vector may be one which,when introduced into a cell, is integrated into the genome of therecipient cell and replicated together with the chromosome(s) into whichit has been integrated. A vector system may comprise a single vector orplasmid, two or more vectors or plasmids, which together contain thetotal DNA to be introduced into the genome of the host cell, or atransposon. The choice of the vector will typically depend on thecompatibility of the vector with the cell into which the vector is to beintroduced. The vector may also include a selection marker such as anantibiotic resistance gene that can be used for selection of suitabletransformants, or sequences that enhance transformation of prokaryoticor eukaryotic (especially wheat) cells such as T-DNA or P-DNA sequences.Examples of such resistance genes and sequences are well known to thoseof skill in the art.

By “marker gene” is meant a gene that imparts a distinct phenotype tocells expressing the marker gene and thus allows such transformed cellsto be distinguished from cells that do not have the marker. A“selectable marker gene” confers a trait for which one can ‘select’based on resistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells) orbased on a growth advantage in the presence of a metabolizablesubstrate. A screenable marker gene (or reporter gene) confers a traitthat one can identify through observation or testing, i.e., by‘screening’ (e.g., β-glucuronidase, luciferase, GFP or other enzymeactivity not present in untransformed cells). The marker gene and thenucleotide sequence of interest do not have to be linked.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, kanamycin, erythromycin,chloramphenicol or tetracycline resistance. Exemplary selectable markersfor selection of plant transformants include, but are not limited to, ahyg gene which confers hygromycin B resistance; a neomycinphosphotransferase (npt) gene conferring resistance to kanamycin,paromomycin, G418 and the like as, for example, described by Potrykus etal., 1985; a glutathione-S-transferase gene from rat liver conferringresistance to glutathione derived herbicides as, for example, describedin EP-A-256223; a glutamine synthetase gene conferring, uponoverexpression, resistance to glutamine synthetase inhibitors such asphosphinothricin as, for example, described WO87/05327, an acetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP-A-275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al., 1988, a bar gene conferringresistance against bialaphos as, for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal, 1988); a mutant acetolactate synthase gene (ALS), which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (EP-A-154204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (GFP, Niedzet al., 1995) or one of its variants; a luciferase (luc) gene (Ow etal., 1986), which allows for bioluminescence detection, and others knownin the art.

In some embodiments, the level of endogenous starch branching activityor other enzyme activity is modulated by decreasing the level ofexpression of genes encoding proteins involved in these activities inthe wheat plant, or increasing the level of expression of a nucleotidesequence that codes for the enzyme in a wheat plant. Increasingexpression can be achieved at the level of transcription by usingpromoters of different strengths or inducible promoters, which arecapable of controlling the level of transcript expressed from the codingsequence. Heterologous sequences may be introduced which encodetranscription factors that modulate or enhance expression of genes whoseproducts down regulate starch branching. The level of expression of thegene may be modulated by altering the copy number per cell of aconstruct comprising the coding sequence and a transcriptional controlelement that is operably connected thereto and that is functional in thecell. Alternatively, a plurality of transformants may be selected, andscreened for those with a favourable level and/or specificity oftransgene expression arising from influences of endogenous sequences inthe vicinity of the transgene integration site. A favourable level andpattern of transgene expression is one which results in a substantialincrease in starch synthesis or amylose content in the wheat plant. Thismay be detected by simple testing of transformants.

Reducing gene expression may be achieved through introduction andtranscription of a “gene-silencing chimeric gene” introduced into thewheat plant. The gene-silencing chimeric gene is preferably introducedstably into the wheat genome, preferably the wheat nuclear genome. Asused herein “gene-silencing effect” refers to the reduction ofexpression of a target nucleic acid in a wheat cell, preferably anendosperm cell, which can be achieved by introduction of a silencingRNA. In a preferred embodiment, a gene-silencing chimeric gene isintroduced which encodes an RNA molecule which reduces expression of oneor more endogenous genes, preferably the SBEIIa and/or SBEIIb genes.Target genes in wheat also include the genes listed in Table 1. Suchreduction may be the result of reduction of transcription, including viamethylation of chromatin remodeling, or post-transcriptionalmodification of the RNA molecules, including via RNA degradation, orboth. Gene-silencing should not necessarily be interpreted as anabolishing of the expression of the target nucleic acid or gene. It issufficient that the level expression of the target nucleic acid in thepresence of the silencing RNA is lower that in the absence thereof. Thelevel of expression of the targeted gene may be reduced by at leastabout 40% or at least about 45% or at least about 50% or at least about55% or at least about 60% or at least about 65% or at least about 70% orat least about 75% or at least about 80% or at least about 85% or atleast about 90% or at least about 95% or effectively abolished to anundetectable level.

Antisense techniques may be used to reduce gene expression in wheatcells. The term “antisense RNA” shall be taken to mean an RNA moleculethat is complementary to at least a portion of a specific mRNA moleculeand capable of reducing expression of the gene encoding the mRNA,preferably a SBEIIa and/or SBEIIb gene. Such reduction typically occursin a sequence-dependent manner and is thought to occur by interferingwith a post-transcriptional event such as mRNA transport from nucleus tocytoplasm, mRNA stability or inhibition of translation. The use ofantisense methods is well known in the art (see for example, Hartmannand Endres, 1999). Antisense methods are now a well establishedtechnique for manipulating gene expression in plants.

Antisense molecules typically include sequences that correspond to partof the transcribed region of a target gene or for sequences that effectcontrol over the gene expression or splicing event. For example, theantisense sequence may correspond to the targeted protein coding regionof the genes of the invention, or the 5′-untranslated region (UTR) orthe 3′-UTR or combination of these, preferably only to exon sequences ofthe target gene. In view of the generally greater divergence betweenrelated genes of the UTRs, targeting these regions provides greaterspecificity of gene inhibition. The length of the antisense sequenceshould be at least 19 contiguous nucleotides, preferably at least 50nucleotides, and more preferably at least 100, 200, 500 or 1000nucleotides, to a maximum of the full length of the gene to beinhibited. The full-length sequence complementary to the entire genetranscript may be used. The length is most preferably 100-2000nucleotides. The degree of identity of the antisense sequence to thetargeted transcript should be at least 90% and more preferably 95-100%.The antisense RNA molecule may of course comprise unrelated sequenceswhich may function to stabilize the molecule.

Genetic constructs to express an antisense RNA may be readily made byjoining a promoter sequence to a region of the target gene in an“antisense” orientation, which as used herein refers to the reverseorientation relative to the orientation of transcription and translation(if it occurs) of the sequence in the target gene in the plant cell.Preferably, the antisense RNA is expressed preferentially in theendosperm of a wheat plant by use of an endosperm-specific promoter.

The term “ribozyme” refers to an RNA molecule which specificallyrecognizes a distinct substrate RNA and catalyzes its cleavage.Typically, the ribozyme contains an antisense sequence for specificrecognition of a target nucleic acid, and an enzymatic region referredto herein as the “catalytic domain”. The types of ribozymes that areparticularly useful in this invention are the hammerhead ribozyme(Haseloff and Gerlach, 1988; Perriman et al., 1992) and the hairpinribozyme (Shippy et al., 1999).

As used herein, “artificially introduced dsRNA molecule” refers to theintroduction of double-stranded RNA (dsRNA) molecule, which preferablyis synthesised in the wheat cell by transcription from a chimeric geneencoding such dsRNA molecule. RNA interference (RNAi) is particularlyuseful for specifically reducing the expression of a gene or inhibitingthe production of a particular protein, also in wheat (Regina et al.,2006). This technology relies on the presence of dsRNA molecules thatcontain a sequence that is essentially identical to the mRNA of the geneof interest or part thereof, and its complement, thereby forming adsRNA. Conveniently, the dsRNA can be produced from a single promoter inthe host cell, where the sense and anti-sense sequences are transcribedto produce a hairpin RNA in which the sense and anti-sense sequenceshybridize to form the dsRNA region with a related (to a SBEII gene) orunrelated sequence forming a loop structure, so the hairpin RNAcomprises a stem-loop structure. The design and production of suitabledsRNA molecules for the present invention is well within the capacity ofa person skilled in the art, particularly considering Waterhouse et al.,1998; Smith et al., 2000; WO 99/32619; WO 99/53050; WO 99/49029; and WO01/34815.

The DNA encoding the dsRNA typically comprises both sense and antisensesequences arranged as an inverted repeat. In a preferred embodiment, thesense and antisense sequences are separated by a spacer region thatcomprises an intron which, when transcribed into RNA, is spliced out.This arrangement has been shown to result in a higher efficiency of genesilencing (Smith et al., 2000). The double-stranded region may compriseone or two RNA molecules, transcribed from either one DNA region or two.The dsRNA may be classified as long hpRNA, having long, sense andantisense regions which can be largely complementary, but need not beentirely complementary (typically larger than about 200 bp, rangingbetween 200-1000 bp). hpRNA can also be rather small with thedouble-stranded portion ranging in size from about 30 to about 42 bp,but not much longer than 94 bp (see WO04/073390). The presence of thedouble stranded RNA region is thought to trigger a response from anendogenous plant system that destroys both the double stranded RNA andalso the homologous RNA transcript from the target plant gene,efficiently reducing or eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridise shouldeach be at least 19 contiguous nucleotides, preferably at least 30 or 50nucleotides, and more preferably at least 100, 200, 500 or 1000nucleotides. The full-length sequence corresponding to the entire genetranscript may be used. The lengths are most preferably 100-2000nucleotides. The degree of identity of the sense and antisense sequencesto the targeted transcript should be at least 85%, preferably at least90% and more preferably 95-100%. The longer the sequence, the lessstringent the requirement for the overall sequence identity. The RNAmolecule may of course comprise unrelated sequences which may functionto stabilize the molecule. The promoter used to express thedsRNA-forming construct may be any type of promoter that is expressed inthe cells which express the target gene. When the target gene is SBEIIaor SBEIIb or other gene expressed selectively in the endosperm, anendosperm promoter is preferred, so as to not affect expression of thetarget gene(s) in other tissues.

Examples of dsRNA molecules that may be used to down-regulate SBEIIgene(s) are provided in Example 4.

Other silencing RNA may be “unpolyadenylated RNA” comprising at least 20consecutive nucleotides having at least 95% sequence identity to thecomplement of a nucleotide sequence of an RNA transcript of the targetgene, such as described in WO01/12824 or U.S. Pat. No. 6,423,885. Yetanother type of silencing RNA is an RNA molecule as described inWO03/076619 (herein incorporated by reference) comprising at least 20consecutive nucleotides having at least 95% sequence identity to thesequence of the target nucleic acid or the complement thereof, andfurther comprising a largely-double stranded region as described inWO03/076619.

As used herein, “silencing RNAs” are RNA molecules that have 21 to 24contiguous nucleotides that are complementary to a region of the mRNAtranscribed from the target gene, preferably SBEIIa or SBEIIb. Thesequence of the 21 to 24 nucleotides is preferably fully complementaryto a sequence of 21 to 24 contiguous nucleotides of the mRNA i.e.identical to the complement of the 21 to 24 nucleotides of the region ofthe mRNA. However, miRNA sequences which have up to five mismatches inregion of the mRNA may also be used (Palatnik et al., 2003), andbasepairing may involve one or two G-U basepairs. When not all of the 21to 24 nucleotides of the silencing RNA are able to basepair with themRNA, it is preferred that there are only one or two mismatches betweenthe 21 to 24 nucleotides of the silencing RNA and the region of themRNA. With respect to the miRNAs, it is preferred that any mismatches,up to the maximum of five, are found towards the 3′ end of the miRNA. Ina preferred embodiment, there are not more than one or two mismatchesbetween the sequences of the silencing RNA and its target mRNA.

Silencing RNAs derive from longer RNA molecules that are encoded by thechimeric DNAs of the invention. The longer RNA molecules, also referredto herein as “precursor RNAs”, are the initial products produced bytranscription from the chimeric DNAs in the wheat cells and havepartially double-stranded character formed by intra-molecularbasepairing between complementary regions. The precursor RNAs areprocessed by a specialized class of RNAses, commonly called “Dicer(s)”,into the silencing RNAs, typically of 21 to 24 nucleotides long.Silencing RNAs as used herein include short interfering RNAs (siRNAs)and microRNAs (miRNAs), which differ in their biosynthesis. SiRNAsderive from fully or partially double-stranded RNAs having at least 21contiguous basepairs, including possible G-U basepairs, withoutmismatches or non-basepaired nucleotides bulging out from thedouble-stranded region. These double-stranded RNAs are formed fromeither a single, self-complementary transcript which forms by foldingback on itself and forming a stem-loop structure, referred to herein asa “hairpin RNA”, or from two separate RNAs which are at least partlycomplementary and that hybridize to form a double-stranded RNA region.MiRNAs are produced by processing of longer, single-stranded transcriptsthat include complementary regions that are not fully complementary andso form an imperfectly basepaired structure, so having mismatched ornon-basepaired nucleotides within the partly double-stranded structure.The basepaired structure may also include G-U basepairs. Processing ofthe precursor RNAs to form miRNAs leads to the preferential accumulationof one distinct, small RNA having a specific sequence, the miRNA. It isderived from one strand of the precursor RNA, typically the “antisense”strand of the precursor RNA, whereas processing of the longcomplementary precursor RNA to form siRNAs produces a population ofsiRNAs which are not uniform in sequence but correspond to many portionsand from both strands of the precursor.

MiRNAs were first discovered as a small regulatory RNA controlling thelin-4 gene in C. elegans (Lee et al., 1993). Since then, large numbersof other naturally occurring miRNAs have been reported to be involved inregulation of gene function in animals and plants. MiRNA precursor RNAsof the invention, also termed herein as “artificial miRNA precursors”,are typically derived from naturally occurring miRNA precursors byaltering the nucleotide sequence of the miRNA portion of thenaturally-occurring precursor so that it is complementary, preferablyfully complementary, to the 21 to 24 nucleotide region of the targetmRNA, and altering the nucleotide sequence of the complementary regionof the miRNA precursor that basepairs to the miRNA sequence to maintainbasepairing. The remainder of the miRNA precursor RNA may be unalteredand so have the same sequence as the naturally occurring miRNAprecursor, or it may also be altered in sequence by nucleotidesubstitutions, nucleotide insertions, or preferably nucleotidedeletions, or any combination thereof. The remainder of the miRNAprecursor RNA is thought to be involved in recognition of the structureby the Dicer enzyme called Dicer-like 1 (DCL1), and therefore it ispreferred that few if any changes are made to the remainder of thestructure. For example, basepaired nucleotides may be substituted forother basepaired nucleotides without major change to the overallstructure. The naturally occurring miRNA precursor from which theartificial miRNA precursor of the invention is derived may be fromwheat, another plant such as another cereal plant, or from non-plantsources. Examples of such precursor RNAs are the rice mi395 precursor,the Arabidopsis mi159b precursor, or the mi172 precursor.

Artificial miRNAs have been demonstrated in plants, for example Alvarezet al., 2006; Parizotto et al., 2004; Schwab et al., 2006.

Another molecular biological approach that may be used isco-suppression. The mechanism of co-suppression is not well understoodbut is thought to involve post-transcriptional gene silencing (PTGS) andin that regard may be very similar to many examples of antisensesuppression. It involves introducing an extra copy of a gene or afragment thereof into a plant in the “sense orientation” with respect toa promoter for its expression, which as used herein refers to the sameorientation as transcription and translation (if it occurs) of thesequence relative to the sequence in the target gene. The size of thesense fragment, its correspondence to target gene regions, and itsdegree of homology to the target gene are as for the antisense sequencesdescribed above. In some instances the additional copy of the genesequence interferes with the expression of the target plant gene.Reference is made to Patent specification WO 97/20936 and Europeanpatent specification 0465572 for methods of implementing co-suppressionapproaches. The antisense, co-suppression or double stranded RNAmolecules may also comprise a largely double-stranded RNA region,preferably comprising a nuclear localization signal, as described in WO03/076619.

Any of these technologies for reducing gene expression can be used tocoordinately reduce the activity of multiple genes. For example, one RNAmolecule can be targeted against a family of related genes by targetinga region of the genes which is in common. Alternatively, unrelated genesmay be targeted by including multiple regions in one RNA molecule, eachregion targeting a different gene. This can readily be done by fusingthe multiple regions under the control of a single promoter.

A number of techniques are available for the introduction of nucleicacid molecules into a wheat cell, well known to workers in the art. Theterm “transformation” as used herein means alteration of the genotype ofa cell, for example a bacterium or a plant, particularly a wheat plant,by the introduction of a foreign or exogenous nucleic acid. By“transformant” is meant an organism so altered. Introduction of DNA intoa wheat plant by crossing parental plants or by mutagenesis per se isnot included in transformation. As used herein the term “transgenic”refers to a genetically modified plant in which the endogenous genome issupplemented or modified by the random or site-directed integration, orstable maintenance in a replicable non-integrated form, of an introducedforeign or exogenous gene or sequence. By “transgene” is meant a foreignor exogenous gene or sequence that is introduced into a plant. Thenucleic acid molecule may be replicated as an extrachromosomal elementor is preferably stably integrated into the genome of the plant. By“genome” is meant the total inherited genetic complement of the cell,plant or plant part, and includes chromosomal DNA, plastid DNA,mitochondrial DNA and extrachromosomal DNA molecules. In an embodiment,a transgene is integrated in the wheat nuclear genome which in hexaploidwheat includes the A, B and D subgenomes, herein referred to as the A, Band D “genomes”.

The most commonly used methods to produce fertile, transgenic wheatplants comprise two steps: the delivery of DNA into regenerable wheatcells and plant regeneration through in vitro tissue culture. Twomethods are commonly used to deliver the DNA: T-DNA transfer usingAgrobacterium tumefaciens or related bacteria and direct introduction ofDNA via particle bombardment, although other methods have been used tointegrate DNA sequences into wheat or other cereals. It will be apparentto the skilled person that the particular choice of a transformationsystem to introduce a nucleic acid construct into plant cells is notessential to or a limitation of the invention, provided it achieves anacceptable level of nucleic acid transfer. Such techniques for wheat arewell known in the art.

Transformed wheat plants can be produced by introducing a nucleic acidconstruct according to the invention into a recipient cell and growing anew plant that comprises and expresses a polynucleotide according to theinvention. The process of growing a new plant from a transformed cellwhich is in cell culture is referred to herein as “regeneration”.Regenerable wheat cells include cells of mature embryos, meristematictissue such as the mesophyll cells of the leaf base, or preferably fromthe scutella of immature embryos, obtained 12-20 days post-anthesis, orcallus derived from any of these. The most commonly used route torecover regenerated wheat plants is somatic embryogenesis using mediasuch as MS-agar supplemented with an auxin such as 2,4-D and a low levelof cytokinin, see Sparks and Jones, 2004).

Agrobacterium-mediated transformation of wheat may be performed by themethods of Cheng et al., 1997; Weir et al., 2001; Kanna and Daggard,2003 or Wu et al., 2003. Any Agrobacterium strain with sufficientvirulence may be used, preferably strains having additional virulencegene functions such as LBA4404, AGL0 or AGL1 (Lazo et al., 1991) orversions of C58. Bacteria related to Agrobacterium may also be used. TheDNA that is transferred (T-DNA) from the Agrobacterium to the recipientwheat cells is comprised in a genetic construct (chimeric plasmid) thatcontains one or two border regions of a T-DNA region of a wild-type Tiplasmid flanking the nucleic acid to be transferred. The geneticconstruct may contain two or more T-DNAs, for example where one T-DNAcontains the gene of interest and a second T-DNA contains a selectablemarker gene, providing for independent insertion of the two T-DNAs andpossible segregation of the selectable marker gene away from thetransgene of interest.

Any wheat type that is regenerable may be used; varieties Bob White,Fielder, Veery-5, Cadenza and Florida have been reported with success.Transformation events in one of these more readily regenerable varietiesmay be transferred to any other wheat cultivars including elitevarieties by standard backcrossing. An alternative method usingAgrobacterium makes use of an in vivo inoculation method followed byregeneration and selection of transformed plants using tissue cultureand has proven to be efficient, see WO00/63398. Other methods involvingthe use of Agrobacterium include: co-cultivation of Agrobacterium withcultured isolated protoplasts; transformation of seeds, apices ormeristems with Agrobacterium, or inoculation in planta such as thefloral-dip method for Arabidopsis as described by Bechtold et al., 1993.This latter approach is based on the vacuum infiltration of a suspensionof Agrobacterium cells. Alternatively, the chimeric construct may beintroduced using root-inducing (Ri) plasmids of Agrobacterium asvectors.

Another method commonly used for introducing the nucleic acid constructinto a plant cell is high velocity ballistic penetration by smallparticles (also known as particle bombardment or microprojectilebombardment) with the nucleic acid to be introduced contained eitherwithin the matrix of small beads or particles, or on the surface thereofas, for example described by Klein et al., 1987. This method has beenadapted for wheat (Vasil, 1990). Microprojectile bombardment to inducewounding followed by co-cultivation with Agrobacterium may be used(EP-A-486233). The genetic construct can also be introduced into plantcells by electroporation as, for example, described by Fromm et al.,1985 and Shimamoto et al., 1989. Alternatively, the nucleic acidconstruct can be introduced into a wheat cell such as a pollen cell bycontacting the cell with the nucleic acid using mechanical or chemicalmeans.

Preferred selectable marker genes for use in the transformation of wheatinclude the Streptomyces hygroscopicus bar gene or pat gene inconjunction with selection using the herbicide glufosinate ammonium, thehpt gene in conjunction with the antibiotic hygromycin, or the nptIIgene with kanamycin or G418. Alternatively, positively selectablemarkers such as the manA gene encoding phosphomannose isomerase (PMI)with the sugar mannose-6-phosphate as sole C source may be used.

The present invention is further described by the following non-limitingExamples.

Example 1: Methods and Materials

Carbohydrate Determination and Analysis.

Starch was isolated on small scale from both developing and mature wheatgrain using the method of Regina et al., (2006). Large scale starchextraction was carried out following the method of Regina et al.,(2004). Starch content was determined using the total starch analysiskit supplied by Megazyme (Bray, Co Wicklow, Republic of Ireland) andcalculated on a weight basis as a percentage of the mature, unmilledgrain weight. The starch content was then compared to control plants.Subtraction of the starch weight from the total grain weight to give atotal non-starch content of the grain determined whether the reductionin total weight was due to a reduction in starch content.

The amylose content of starch samples was determined by the colorimetric(iodometric) method of Morrison and Laignelet (1983) with slightmodifications as follows. Approximately 2 mg of starch was weighedaccurately (accurate to 0.1 mg) into a 2 ml screw-capped tube fittedwith a rubber washer in the lid. To remove lipid, 1 ml of 85% (v/v)methanol was mixed with the starch and the tube heated in a 65° C. waterbath for 1 hour with occasional vortexing. After centrifugation at13,000 g for 5 min, the supernatant was carefully removed and theextraction steps repeated. The starch was then dried at 65° C. for 1hour and dissolved in urea-dimethyl sulphoxide solution (UDMSO; 9volumes of dimethyl sulphoxide to 1 volume of 6 M urea), using 1 ml ofUDMSO per 2 mg of starch (weighed as above). The mixture was immediatelyvortexed vigorously and incubated in a 95° C. water bath for 1 hour withintermittent vortexing for complete dissolution of the starch. Analiquot of the starch-UDMSO solution (50 μl) was treated with 20 μl ofI₂-KI reagent that contained 2 mg iodine and 20 mg potassium iodide perml of water. The mixture was made up to 1 ml with water. The absorbanceof the mixture at 620 nm was measured by transferring 200 μl tomicroplate and reading the absorbance using an Emax Precision MicroplateReader (Molecular Devices, USA). Standard samples containing from 0 to100% amylose and 100% to 0% amylopectin were made from potato amyloseand corn (or potato) amylopectin (Sigma) and treated as for the testsamples. The amylose content (percentage amylose) was determined fromthe absorbance values using a regression equation derived from theabsorbances for the standard samples. Analysis of theamylose/amylopectin ratio of non-debranched starches may also be carriedout according to Case et al., (1998) or by an HPLC method using 90% DMSOfor separating debranched starches as described by Batey and Curtin,(1996).

Statistical analysis of the amylose data was carried out using the8^(th) edition of Genstat for Windows (VSN International Ltd, Herts,UK).

The distribution of chain lengths in the starch was analysed byfluorophore assisted carbohydrate electrophoresis (FACE) using acapillary electrophoresis unit according to Morell et al., (1998) afterdebranching of the starch samples. The gelatinisation temperatureprofiles of starch samples were measured in a Pyris 1 differentialscanning calorimeter (Perkin Elmer, Norwalk Conn., USA). The viscosityof starch solutions was measured on a Rapid-Visco-Analyser (RVA, NewportScientific Pty Ltd, Warriewood, Sydney), for example using conditions asreported by Batey et al., (1997). The parameters measured included peakviscosity (the maximum hot paste viscosity), holding strength, finalviscosity and pasting temperature. The swelling volume of flour orstarch was determined according to the method of Konik-Rose et al.,(2001). The uptake of water was measured by weighing the sample prior toand after mixing the flour or starch sample in water at definedtemperatures and following collection of the gelatinized material.

Starch granule morphology was analysed by microscopy. Purified starchgranule suspensions in water were examined under both normal andpolarized light using a Leica-DMR compound microscope to determine thestarch granule morphology. Scanning electron microscopy was carried outusing a Joel JSM 35C instrument. Purified starches were sputter-coatedwith gold and scanned at 15 kV at room temperature.

β-Glucan levels were determined using the kit supplied by Megazyme(Bray, Co, Wicklow, Republic of Ireland).

Analysis of Protein Expression in Endosperm.

Specific expression of SBEI, SBEIIa and SBEIIb proteins in endosperm, inparticular the level of expression or accumulation of these proteins,was analysed by Western blot procedures. Endosperm was dissected awayfrom all maternal tissues and samples of approximately 0.2 mg werehomogenized in 600 μl of 50 mM K phosphate buffer (42 mM K₂HPO₄ and 8 mMKH₂PO₄), pH 7.5, containing 5 mM EDTA, 20% glycerol, 5 mM DTT and 1 mMPefabloc. The ground samples were centrifuged for 10 min at 13,000 g andthe supernatant aliquoted and frozen at −80° C. until use. For totalprotein estimation, a BSA standard curve was set up using 0, 20, 40, 60,80 and 100 μl aliquots of 0.25 mg/ml BSA standard. The samples (3 μl)were made up to 100 μl with distilled water and 1 ml of Coomassie PlusProtein reagent was added to each. The absorbance was read after 5 minat 595 nm, using the zero BSA sample from the standard curve as theblank, and the protein levels in the samples determined. Samplescontaining 20 μg total protein from each endosperm were run on an 8% nondenaturing polyacrylamide gel containing 0.34 M Tris-HCl (pH 8.8),acrylamide (8.0%), ammonium persulphate (0.06%) and TEMED (0.1%).Following electrophoresis, the proteins were transferred to anitrocellulose membrane according to Morell et al., 1997 andimmunoreacted with SBEIIa, SBEIIb or SBEI specific antibodies. Antiserumagainst wheat SBEIIa protein (anti-wBEIIa) was generated using asynthetic peptide having the amino acid sequence of the N-terminalsequence of mature wheat SBEIIa, AASPGKVLVPDGESDDL (SEQ ID NO: 16)(Rahman et al., 2001). Antiserum against wheat SBEIIb (anti-wBEIIb) wasgenerated in an analogous manner using the N-terminal synthetic peptide,AGGPSGEVMI (SEQ ID NO: 17) (Regina et al., (2005). This peptide wasthought to represent the N-terminal sequence of the mature SBEIIbpeptide and furthermore was identical to the N-terminus of the barleySBEIIb protein (Sun et al., 1998). A polyclonal antibody against wheatSBEI was synthesised in an analogous manner using the N-terminalsynthetic peptide VSAPRDYTMATAEDGV (SEQ ID NO: 18) (Morell et al.,1997). Such antisera were obtained from rabbits immunised with thesynthetic peptides according to standard methods.

Enzyme Assay for SBE.

Enzyme activity assays of branching enzymes to detect the activity ofall three isoforms, SBEI, SBEIIa and SBEIIb was based on the method ofNishi et al., 2001 with minor modification. After electrophoresis, thegel was washed twice in 50 mM HEPES, pH 7.0 containing 10% glycerol andincubated at room temperature in a reaction mixture consisting of 50 mMHEPES, pH 7.4, 50 mM glucose-1-phosphate, 2.5 mM AMP, 10% glycerol, 50 Uphosphorylase a 1 mM DTT and 0.08% maltotriose for 16 h. The bands werevisualised with a solution of 0.2% (W/V) 12 and 2% KI. The SBEI, SBEIIaand SBEIIb isoform specific activities were separated under theseconditions of electrophoresis. This was confirmed by immunoblottingusing anti-SBEI, anti-SBEIIa and anti-SBEIIb antibodies. Densitometricanalysis of immunoblots using TotalLab software package (NonlinearDynamics Ltd, Newcastle, UK) which measures the intensity of each bandwas conducted to determine the level of enzyme activity of each isoform.

Starch branching enzyme (SBE) activity may be measured by enzyme assay,for example by the phosphorylase stimulation assay (Boyer and Preiss,1978). This assay measures the stimulation by SBE of the incorporationof glucose 1-phosphate into methanol-insoluble polymer (α-D-glucan) byphosphorylase A. Activity of specific isoforms of SBE can be measured bythis assay following purification of individual isoforms as described inRegina et al., 2004. The total soluble protein extracts were applied toa 3 ml β-cyclodextrin 03-CD) affinity column pre-equilibrated with theextraction buffer described above. The column was prepared by couplingβ-CD to Epoxy-activated sepharose 6B (Amersham Biosciences, Uppsala,Sweden) following the manufacturer's instructions. The bound proteins(containing SBEs) were eluted using 1% β-CD in Phosphate buffer and thendialysed against buffer A (20 mM phosphate buffer, pH 8.0, 1 mM EDTA and1 mM DTT). The dialysed samples were subjected to anion exchangechromatography using a 1 ml MonoQ column (Amersham Pharmacia),pre-equilibrated with buffer A. After elution of the unbound proteins, a30 min linear gradient was applied by introducing buffer B (500 mMPhosphate buffer, pH 8.0, 1 mM EDTA, 1 mM DTT) into buffer A to elutethe bound proteins.

SBE activity can also be measured by the iodine stain assay, whichmeasures the decrease in the absorbency of a glucan-polyiodine complexresulting from branching of glucan polymers. SBE activity can also beassayed by the branch linkage assay which measures the generation ofreducing ends from reduced amylose as substrate, following isoamylasedigestion (Takeda et al., 1993a). Preferably, the activity is measuredin the absence of SBEI activity. Isoforms of SBE show differentsubstrate specificities, for example SBEI exhibits higher activity inbranching amylose, while SBEIIa and SBEIIb show higher rates ofbranching with an amylopectin substrate. The isoforms may also bedistinguished on the basis of the length of the glucan chain that istransferred. SBE protein may also be measured by using specificantibodies such as those described herein. Preferably, the SBEIIactivity is measured during grain development in the developingendosperm. SBEII protein levels are preferably measured in the maturegrain where the protein is still present by immunological methods suchas Western blot analysis.

DNA Analysis of Wheat Plants.

PCR analysis of transformed wheat plants or of plants to be tested forthe presence of transgenes was performed on genomic DNA extracted from1-2 cm² of fresh leaf material using the mini-prep method described byStacey and Isaac, (1994). PCR assays to determine the presence of thehairpin RNA constructs used the primers SBEIIa-For:5′-CCCGCTGCTTTCGCTCATTTTG-3′ (SEQ ID NO: 19) and SBEIIa-Rev:5′-GACTACCGGAGCTCCCACCTTC-3′ (SEQ ID NO: 20) designed to amplify afragment (462 bp) from the SBEIIa gene. Reaction conditions were asfollows: “hot start” (94° C., 3 min) followed by 30 cycles ofdenaturation (95° C., 30 sec), annealing (55° C., 30 sec), extension(73° C., 2 min) followed by 1 cycle at 73° C. (5 min). Reaction productswere analysed by agarose or polyacrylamide gel electrophoresis.

Southern blot hybridization analysis was performed on DNA from a largerscale (9 ml) extraction from lyophilized ground tissue (Stacey andIsaac, 1994). DNA samples were adjusted to 0.2 mg/ml and digested withrestriction enzymes such as HindIII, EcoRI and KpnI. Restriction enzymedigestion, gel electrophoresis and vacuum blotting are carried out asdescribed by Stacey and Isaac, (1994). Digoxygenin-labelled probesincluding the intron 3 region of the ds-SBEII constructs are produced byPCR according to the method of McCreery and Helentjaris, (1994).Hybridization of the probes to the Southern blot and detection bychemiluminescence are performed according to the method of McCreery andHelentjaris, (1994).

Transformation of Wheat by Agrobacterium.

Genetic constructs for transformation of wheat were introduced byelectroporation into the disarmed Agrobacterium tumefaciens strainLBA4404 carrying the vir plasmid pAL4404 and pSB1, with subsequentselection on media with spectinomycin. Transformed Agrobacterium strainswere incubated on solidified YEP media at 27° C. for 2 days. Bacteriawere then collected and re-suspended in TSIM1 (MS media with 100 mg/lmyo-inositol, 10 g/l glucose, 50 mg/l MES buffer pH5.5) containing 400mM acetosyringone to an optical density of 2.4 at 650 nm for wheatinoculation.

Wheat plants (variety NB1, a Spring wheat variety obtained fromNickerson Seeds Ltd, Rothwell, Lincs.) were grown in a glasshouse at22/15° C. day/night temperature with supplemented light to give a 16hour day. Tillers were harvested approximately 14 days post-anthesis(embryos approximately 1 mm in length) to include 50 cm tiller stem. Allleaves were then removed from the tillers except the flag leaf, whichwas cleaned to remove contaminating fungal spores. The glumes of eachspikelet and the lemma from the first two florets were then carefullyremoved to expose the immature seed. Generally, only these two seed ineach spikelet were uncovered. This procedure was carried out along theentire length of the inflorescence. The ears were then sprayed with 70%IMS as a brief surface sterilization.

Agrobacterium suspensions (1 μl) were inoculated using a 10 μl Hamiltonsyringe into the immature seed approximately at the position of thescutellum:endosperm interface so that all exposed seed were inoculated.The tillers were then placed in water, covered with a translucentplastic bag to prevent seed dehydration, and placed in a lit incubatorfor 3 days at 23° C., 16 hr day, 45 μEm⁻²s⁻¹PAR. After 3 days ofco-cultivation, the inoculated immature seed were removed and surfacesterilized with 70% ethanol (30 sec), then 20% bleach (Domestos, 20min), followed by thorough washing in sterile distilled water. Immatureembryos were aseptically isolated and placed on W3 media (MSsupplemented with 20 g/l sucrose and 2 mg/l 2,4-D and solidified with 6g/l Type I agarose, Sigma) with the addition of 150 mg/l Timentin (W3Tmedium) and with the scutellum uppermost (20 embryos per plate).Cultures were placed at 25° C. in the light (16 hour day, 80μEm⁻²s⁻¹PAR). The development of the embryonic axis on the embryos wasassessed about 5 days after isolation and the axis was removed wherenecessary to improve callus production. The embryos were maintained onW3T for 4 weeks, with a transfer to fresh media at 2 weekspost-isolation and assessed for embryogenic capacity.

After 4 weeks growth, callus derived from the inoculated embryos wasvery similar to control callus obtained from uninoculated embryos platedon W3T medium. Presence of the bacteria did not appear to havesubstantially reduced the embryogenic capacity of the callus derivedfrom the inoculated embryos. Embryogenic calli were transferred to W3media with 2 mg/l Asulam or geneticin at 25 mg/l and 150 mg/l Timentin(W32AT medium). Calli were maintained on this media for a further 2weeks and then each callus was divided into 2 mm-sized pieces andre-plated onto W32AT. Control embryos derived from inoculations with theLBA4404 without binary vector constructs did not produce transformedcallus on selection media.

After a further 2 weeks culture, all tissue was assessed for developmentof embryogenic callus: any callus showing signs of continued developmentafter 4 weeks on selection was transferred to regeneration media (RMT-MSwith 40 g/l maltose and 150 mg/l Timentin, pH 5.8, solidified with 6 g/lagarose, Sigma type 1). Shoots were regenerated within 4 weeks on thismedia and then transferred to MS30 with 150 mg/l Timentin for shootelongation and rooting. Juvenile plants were then transferred to soilmixture and kept on a misting bench for two weeks and finallytransferred to a glasshouse.

Alternative Agrobacterium strains such as strain AGL1 or selectablemarkers such as genes encoding hygromycin resistance can also be used inthe method.

Example 2: Inhibition of SBEIIa Genes in Wheat Using Four Hairpin RNAConstructs

Four hairpin RNA (dsRNA) constructs were made to reduce the expressionof i) the SBEIIa, or ii) the SBEIIa, SBEIIb and SBEI genes of wheat. Ineach construct, the DNA encoding the hairpin RNA was linked to a highmolecular weight glutenin (HMWG) promoter sequence obtained from a wheatDx5 gene to provide endosperm-specific expression of the hairpin RNA,and a transcription terminator sequence from the nopaline synthase genefrom Agrobacterium (nos3′). This promoter provided forendosperm-specific expression of the synthetic genes encoding thehairpin RNAs.

hp5′-SBEIIa.

The construction and use of the first of the constructs, designated ashp5′-SBEIIa, is described in Regina et al., (2006). The hp5′-SBEIIaconstruct contained 1536 bp of nucleotide sequence amplified by PCR fromthe wheat SBEIIa gene (GenBank Accession number AF338431). This includeda 468 bp sequence that comprises the whole of exons 1 and 2 and part ofexon 3 (nucleotide positions 1058 to 1336, 1664 to 1761 and 2038 to 2219(that includes nucleotide positions 1 to 578 of Aegilops tauschii cDNAencoding SBEIIa, GenBank accession number AF338431.1) with EcoRI andKpnI restriction sites on either side (fragment 1), a 512 bp sequenceconsisting of part of exons 3 and 4 and the whole of intron 3 of SBEIIa(nucleotide positions 2220 to 2731) with KpnI and SacI sites on eitherside (fragment 2) and a 528 bp fragment consisting of the complete exons1, 2 and 3 of SBEIIa (nucleotide positions 1058 to 1336, 1664 to 1761and 2038 to 2279 in AF338431, that includes nucleotide positions 1 to638 of Aegilops tauschii SBEIIa cDNA, GenBank accession numberAF338431.1) with BamHI and SacI sites on either side (fragment 3).Fragments 1, 2 and 3 were then ligated so that the sequence of fragment3 was ligated to fragment 2 in the antisense orientation relative tofragment 1. The hairpin RNA constructs were initially generated in thevector pDVO3000 which contains the HMWG promoter sequence and nos3′terminator.

hpc-SBEIIa.

The SBEIIa construct designated hpc-SBEIIa comprised a 293 basepair DNAfragment corresponding to nucleotides 1255 to 1547 of the SBEIIa cDNA(GenBank Accession No. AF338432.1), which corresponds to part of exon12, exons 13 and 14 and part of exon 15 of the SBEIIa gene. This regionof SBEIIa was chosen because it had only about 81% identity to thenucleotide sequence of the corresponding region of SBEIIb cDNA, thusincreasing the chance of specificity of silencing of SBEIIa but notSBEIIb.

hp3′-SBEIIa.

The SBEIIa construct designated hp3′-SBEIIa comprised a 130 basepair DNAfragment corresponding to nucleotides 2305 to 2434 of the SBEIIa cDNA,corresponding to part of exon 21, exon 22 and part of the 3′untranslated region (3′ UTR) of the SBEIIa gene.

hp-Combo.

The hairpin RNA construct designated hp-combo comprised regions of thewheat SBEI gene in addition to parts of the SBEIIa gene, and containedi) a 417 basepair sequence corresponding to nucleotides 1756 to 2172from the SBEIIa cDNA, corresponding to part of exon 16, exons 17 to 19,and part of exon 20, and ii) a 357 basepair sequence corresponding tonucleotides 267 to 623 of an SBEI cDNA (GenBank Accession No. AF076679),corresponding to part of exon 3, exon 4, and part of exon 5 of the SBEIgene. The SBEIIa gene fragment had about 86% identity to thecorresponding region of the SBEIIb gene, including several regions of 23consecutive nucleotides with 100% identity to their correspondingregions of SBEIIb, and therefore the combination construct was designedwith the expectation that it would reduce expression of the genesencoding SBEIIb as well as the genes encoding SBEIIa and SBEI in wheat.

Two copies of each of the fragments described above were inserted, onein sense and the other in antisense orientation, into a suitable vector,such that a rice tubulin gene intron was present between the two copies.The synthetic gene was inserted into a binary vector and used totransform wheat.

These constructs were used to transform wheat as described in Example 1.The numbers of independent wheat transgenic lines that were PCR positivefor the respective constructs were as follows: hp5′-SBEIIa, 27;hpc-SBEIIa, 10; hp3′-SBEIIa, 10; and hp-combo, 63.

Analyses of Transgenic Plants: DNA Analysis.

PCR analysis was performed to detect one or more of the transgenes inthe regenerated plants using genomic DNA extracted from 1-2 cm² of freshleaf material using the mini-prep method described by Stacey and Isaac,(1994). PCR reactions were performed for plants transformed with thehp5′-SBEIIa transgene, for example, using the primers SBEIIa-For:5′-CCCGCTGCTTTCGCTCATTTTG-3′ (SEQ ID NO: 19) and SBEIIa-Rev:5′-GACTACCGGAGCTCCCACCTTC-3′ (SEQ ID NO: 20). These PCR reactions weredesigned to amplify a fragment of about 462 bp from the SBEIIa gene.Reaction conditions were as follows: “hot start” (94° C., 3 min)followed by 30 cycles of denaturation (95° C., 30 sec), annealing (55°C., 30 sec) and extension (73° C., 2 min), followed by 1 cycle at 73° C.(5 min).

Starch Granule Morphology.

The morphology of starch granules from mature T1 seed obtained from theT0 transformed wheat plants was observed by light microscopy. Tenindividual grains from each of 25 T0 hp5′-SBEIIa plants were analysed.Each endosperm was gently crushed to release the starch granules whichwere dispersed in water and visualized under a light microscope. Of the25 lines analysed, 12 had grains with distorted granules although thevisual observation revealed varying levels of distortion in differentseeds. Nine seeds from each of the plants transformed with thehpc-SBEIIa, hp3′-SBEIIa and hp-combo transgenes were similarly analysedfor morphological alterations in the starch granules. In this case,half-seeds were analysed so that each remaining halfseed could be growninto a T1 plant, thus preserving each line. Fifty-five out of 63hp-combo lines had seeds with altered granule morphology with varyinglevels of distortion. All of the ten hp5′-SBEIIa lines had seeds withaltered starch granule morphology, again with varying levels ofdistortion. No significant starch granule morphology alteration wasobserved in any of the SBEIIa 3′ lines. Distorted starch granules are anindicator of elevated amylose levels in the starch of the endosperm,typically above 50% amylose, or above 70% amylose for highly distortedstarch granules. This indicated that a range in the extent of thephenotype was observed for each of the effective silencing constructs.

Protein Expression by Western Blotting in Developing Endosperm.

Four to seven T2 developing endosperms from T1 transgenic lines wereanalysed for the level of SBEIIa and SBEIIb proteins by Western blottingusing anti-SBEIIa and anti-SBEIIb antibodies, respectively. In the caseof hp-combo lines, SBEI expression was also analysed using anti-SBEIantibody. Total SBEII protein levels (SBEIIa and SBEIIb) from selectedtransgenic lines were calculated as a percentage of the level in thewild-type (variety NB1) and is shown in Table 11. Amylose levels inmature grain from the transgenic lines, calculated as a percentage ofthe total starch in the grain, was also determined (Table 11) using aniodometric method as described in Example 1. This is representedgraphically in FIG. 5.

A range of expression levels of SBEIIa and SBEIIb were obtained in thegrain of the transgenic plants of independent lines. Such a range isnormally expected in transgenic lines obtained with any one construct,due to the variation in integration sites of the transgene in differenttransgenic events, commonly referred to as “position effect”. The rangeof expression levels seen in these experiments was extended because itwas observed that the four constructs were not equally efficient inreducing the expression of the SBEIIa and SBEIIb genes. In particular,the extent of reduction in the expression of SBEIIb caused by thehp-combo construct in some transformed lines did not correlate with theextent of reduction in expression of SBEIIa, for example lines 679.5.3and 672.2.3. However, all of the constructs reduced expression of thecorresponding genes in a majority of transformed lines.

When the percentage of amylose was plotted against the total SBEIIprotein level and a curve of best fit generated from the data points(see FIG. 5), it was observed that reducing the total SBEII by at least75% relative to the wild-type yielded an amylose content of 50% (w/w) orgreater in the endosperm starch. Reducing the total SBEII activity by atleast 40% relative to the wild-type yielded an amylose content of atleast 40% (w/w).

When the percentage of amylose was plotted against the remaining SBEIIaprotein level, a very similar curve was obtained (see FIG. 6), leadingto the conclusion that the level of SBEIIa in wheat endosperm was theprimary determinant of the amylose level in the starch, and that thelevels of SBEIIb and SBEI were secondary determinants.

The amylose model was further developed based on three sets of inputs(FIG. 6):

-   -   (1) theoretical data based on relative expression levels of        SBEIIa and SBEIIb and amylose data from transgenics    -   (2) amylose data for single and double nulls and theoretical        data based on relative expression levels of SBEIIa and SBEIIb    -   (3) measured amylose data and measured SBEIIa and SBEIIb levels        from the “additional construct” transgenics

In FIG. 6, a power curve has been fitted to this data. Bringing togetherthese three data sets generated a model that was highly consistentbetween input types, reinforcing the model as a predictive tool. Themodel predicted the importance of generating multiple mutations in SBEIIgenes in order to generate high amylose in bread wheat or tetraploidwheat.

Example 3: Cloning and Comparison of SBEII Gene Sequences from Wheat

Isolation of SBEII genes from an Aegilops tauschii genomic library andtheir characterisation by PCR are described in WO99/14314 andWO200162934-A. DNA sequences from the intron 5 region of SBEIIa gene ofthe A, B and D genomes are described in WO200162934-A. Further researchhas led to obtaining sequences from other regions of wheat SBEIIa genesfrom different wheat genotypes and further characterisation of thehomoeologous genes, for example as follows. The exons 12 to 14 region ofSBEIIa was amplified from the hexaploid wheat variety Chara using theprimers AR2aE12F07 (5′-CATTCGTCAAATAATACCCTTGACGG-3′ (SEQ ID NO: 21))and AR2aE14R07 (5′-CTTCACCAATGGATACAGCATCAG-3′ (SEQ ID NO: 22)). Thisyielded a PCR product of about 656 bp which was presumed to be a mixtureof the amplified fragments from each of the three homoeologous genes.This product was sequenced following cloning in a TOPO vector. Threepolymorphic sequences were obtained that covered the region between exon12 to 14 (FIG. 7). Based on PCR analysis of Chinese Spring chromosomeengineered lines using cleavage amplified polymorphic (CAP) markers, thesequence F1-1 was assigned to the D genome, the sequence F1-13 wasassigned to the B genome and the sequence F1-15 was assigned to the Agenome as detailed in Example 4.

The intron 3 region of SBEIIa was amplified from two hexaploid wheatvarieties, Sunco and Tasman, using the primer pair AR2akpnIF(5′-GGTACCGGCAAATATACGAG ATTGACCCG-3′ (SEQ ID NO: 23)) and AR2aSacIR(5′-GAGCTCCCACCTTCATGTT GGTCAATAGC-3′ (SEQ ID NO: 24)). Threepolymorphic sequences were obtained from each of Sunco and Tasman (FIG.8). By comparison with the wheat SBEIIa D genome sequence (GenBankAccession No. AF338431.1), the sequences Tasman 0257 and Sunco 0242 wereassigned to the D genome. Tasman 0272 and Sunco 0241 sequences wereassigned to the B genome based on mapping a polymorphic marker based ona single nucleotide polymorphism in a segregating population. Thesequences Tasman 0264 and Sunco 0243 appeared to be different from the Band D genome sequences and it was concluded they must be from the Agenome. Genotype specific polymorphisms were also observed for thisregion of SBEIIa between Sunco and Tasman in each of the three genomes.

The exon 3 region of SBEIIa from Chinese Spring (CS) was amplified usingthe primers AR2aexon3F (5′-GATACCTGAAGATATCGAGGAGC-3′ (SEQ ID NO: 25))and AR2aexon3R (5′-CGGTAGTCAAGATGGCTCCG-3′ (SEQ ID NO: 26)). Threepolymorphic sequences were obtained (FIG. 9). Comparison with the wheatSBEIIa gene (GenBank Accession No. AF338431.1) revealed that thesequence CS exon 3a was from the D genome. The sequence CS exon 3b wasfound to be from the B genome based on the 100% identity with theGenBank Accession No. FM865435 which was reported to be from a breadwheat 2B chromosome. The third sequence CS exon 3d showed 99% identitywith the GenBank Accession No. Y11282.1, which in turn had a high degreeof identity (99%) with a partial coding sequence reported from the Agenome of Chinese Spring (GenBank Accession No. EU670724). This led tothe prediction that the sequence CS exon 3d was from the A genome.

The exon 1 region of SBEIIa from CS was amplified using the primersAR2aexon1F (5′-CACACGTTGCTCCCCCTTCTC-3′ (SEQ ID NO: 29)) and AR2aexon1R(5′-GAGAGGAGTCCTTCTTCCTGAGG-3′ (SEQ ID NO: 28)). The sequences wereobtained (FIG. 10). Alignment with SBEII GenBank accessions led toassigning the sequence CS exon 1a to the B genome (100% homology toFM865435), CS exon 1b to the A genome (99% homology to Y11282.1) and CSexon 1c to the D genome (100% homology to AF338431.1).

SBEIIa gene sequences were also obtained from the diploid progenitors orrelatives of breadwheat, Triticum urartu which is thought to be the Agenome progenitor of breadwheat, Aegilops speltoides (also known asTriticum speltoides) which is thought to be the B genome progenitor, andAegilops tauschii which is thought to be related closely to the D genomeprogenitor. Gene fragments were obtained from these species as follows:Ten primers were designed based on the nucleotide sequence of the SBEIIagene of the D genome (Accession No. AF338432) or its complement andcovering the whole of that sequence. These primer sets were used toamplify fragments of the SBEIIa genes of diploid species by PCR. Usingthe 10 primers, 16 combinations were used in PCRs with DNA from thediploid species T. urartu (AA genome), A. speltoides (BB), A. tauschii(DD) and the tetraploid species T. durum (AABB genome). In total, 35fragments were selected from these amplifications which were ofsufficient quality for sequencing, to determine their nucleotidesequences. The sequences will be compared and edited using ContigExpress and combined sequences determined for the progenitor SBEIIagenes from the diploids. Polymorphisms such as SNPs orinsertions/deletions will be identified which can be used to distinguishthe genes on the A, B and D genomes, and specific primers designed usingAmplifier for identification of mutants.

The nucleotide sequence of the exon 11-22 region of the SBEIIa gene fromT. urartu is shown in SEQ ID NO: 13, of the exons 3-8 as SEQ ID NO: 15and of exons 1-3 as SEQ ID NO: 14. The nucleotide sequence of the entireSBEIIa gene of A. tauschii is provided in WO2005001098 (incorporatedherein by reference).

Mapping of SBEIIa and SBEIIb-Genetic Linkage of SBEIIa and SBEIIb inWheat.

The SBEIIa and SBEIIb genes were both located on the long arm of wheatchromosome 2 (Regina et al., 2005; Rahman et al., 2001) and based onthese reports were thought to be linked, although it was not knownexactly how close the linkage was. Genetic mapping of the SBEIIa andSBEIIb genes was carried out using a segregating population obtainedfrom a 4-way cross involving the parental cultivars Baxter, Yitpi, Charaand Westonia. The analysis of the population for recombinants betweenthe genes revealed only one recombinant out of approximately 900progeny. From this data, it was calculated that the genetic distancebetween SBEIIa and SBEIIb was only 0.5 cM, which was a very tightlinkage between the two genes.

To determine the physical distance between the two genes, a BAC libraryof Aegilops tauschii constructed by Moullet et al., (1999) was screenedto identifying SBEII containing clones. Hybridisation probes labelledwith ³²P were prepared from the 5′ and 3′ regions from each of theSBEIIa and SBEIIb genes and used to screen the BAC library. Whenscreened with a mixture of the four probes, nine clones were identifiedwith positive hybridisation signals. The nine clones were then screenedseparately with each of the probes and three clones selected. One ofthem (BAC2) was fully sequenced and shown to contain a full lengthSBEIIb gene. Of the other clones, BAC1 was shown to contain a SBEIIagene by partial direct sequencing and BAC3 appeared to contain portionsof both of the SBEIIa and SBEIIb genes as shown by PCR. This indicatedhow closely the two genes are physically linked. BAC1 and BAC3 will befully sequenced. This physical data confirmed the close genetic linkage.

It was therefore predicted that deletion mutations created by agentssuch as radiation which affected one of the genes were likely to extendinto or across both genes i.e. be null for both genes. Furthermore, thissuggested to us the possibility that such deletion mutants might beviable and have wild-type fitness. At least, the observed tight linkageraised the possibility of obtaining mutants with relatively smalldeletions which did not extend to other linked genes needed forviability or fitness. Such mutants were therefore sought as describedbelow in Examples 5-7.

Example 4: Distinguishing the SBEIIa and SBEIIb Homoeologous Genes inWheat

Based on the sequence polymorphisms obtained in Example 2, PCR assayswere designed and prepared to distinguish the homoeologous SBEIIa genesin breadwheat. A nested primer pair, AR2aI13genomeF2(5′-GTACAATTTTACCTGATGAGATCATGG-3′ (SEQ ID NO: 29)) and AR2aI13genomeR2(5′-CTTCAGGAATGGATACAGCATCAG-3′ (SEQ ID NO: 30)) was designed to amplifya 207 bp product from the region between the exons 12 to 14 of wheatSBEIIa. When digested with two restriction enzymes, Ssp1 and Mse1, theproduct amplified using these primers from Chinese Spring (CS) yieldedfour clear bands of sizes 207 bp, 147 bp, 99 bp and 108 bp. Use of thisPCR marker assay on CS chromosome engineered lines revealed that the 207bp product came from the A genome, the 147 bp product came from the Bgenome and the 99 bp and 108 bp products came from the D genome (FIG.11).

Based on SBEIIa sequences from the diploid ancestors of wheat namelyTriticum urartu for genome A, Aegilops speltoides for genome B andAegilops tauschii for genome D, primer pairs were designed that couldspecifically amplify fragments from different regions of the SBEIIagenes from the different genomes and distinguish them (Tables 4 to 8).Tables 6 to 8 list some of the nucleotide polymorphisms (column labelledSNP) and the sizes of the amplified fragments obtained when thedesignated primer pairs are used. These same primer combinations can beused to distinguish the A and B genome homoeologous SBEIIa genes fromdurum wheat.

Development of some PCR primer sets distinguishing the homoeologousSBEIIb genes from the A, B and D genomes of breadwheat and theidentification of SBEIIb in each of these genomes in hexaploid wheat aredescribed in WO200162934-A. Based on SBEIIb sequences from the diploidancestors of wheat namely Triticum urartu for genome A, Aegilopsspeltoides for genome B and Aegilops tauschii for genome D, primer pairsthat could amplify specifically each of the three genomes from differentregions of SBEIIb were designed (Tables 9 to 10). These same primercombinations can be used to distinguish the A and B genome homoeologousSBEIIb genes from durum wheat.

Example 5: Generation and Identification of SBEII Mutants

Mutagenesis of Wheat by Heavy Ion Bombardment.

A mutagenised wheat population was generated in the wheat variety Chara,a commonly used commercial variety, by heavy ion bombardment (HIB) ofwheat seeds. Two sources of heavy ions were used, namely carbon andneon, for mutagenesis which was conducted at Riken Nishina Centre, Wako,Saitama, Japan. Mutagenised seeds were sown in the greenhouse to obtainthe M1 plants. These were selfed to produce the M2 generation. DNAsamples isolated from each of approximately 15,000 M2 plants wereindividually screened for mutations in each of the SBEIIa and SBEIIbgenes using the genome specific PCR primers for SBEIIa (ARIIaF2/ARIIaR2)and SBEIIb (ARA19F/ARA23R) (diagnostic PCR). Each of the PCR reactionson wild-type DNA samples yielded 3 distinct amplification products whichcorresponded to the amplified regions of SBEIIa or SBEIIb genes on theA, B and D genomes, whereas the absence of one of the fragments in thePCRs from mutagenised M2 samples indicated the absence of thecorresponding region in one of the genomes, i.e. the presence of amutant allele in which at least part of the gene was deleted. Suchmutant alleles would almost certainly be null alleles.

Screening of the M2 lines using the genome specific primer pairsidentified a total of 34 mutants which were mutant for the SBEIIa and/orSBEIIb genes. The mutants in SBEIIa were then screened for the presenceof the SBEIIb genes, and vice versa. The identified mutants were therebyclassified into three groups: “Type 1” where both SBEIIa and SBEIIbgenes were mutant i.e. lacking both wild-type genes in one genome, “Type2”, where only the SBEIIa gene was mutant while the SBEIIb gene waswild-type, and “Type 3”, where only the SBEIIb gene was mutant and theSBEIIa gene was wild-type in the particular genome. Since the SBEIIagenes on the A, B and D genomes were distinguished by the diagnostic PCRreactions, and likewise the SBEIIb genes, the mutant alleles could beassigned to one of the genomes according to which amplification productwas absent. As used herein, the designation “A1” refers to the genotypewhere both the SBEIIa and SBEIIb genes on the A genome were mutant, “A2”refers to the genotype where the SBEIIa gene was mutant and the SBEIIbgene on the A genome was wild-type, and “A3” refers to the genotypewhere the SBEIIa gene was wild-type and the SBEIIb gene on the A genomewas mutant. The designations “B1”, “B2”, “B3”, “D1”, “D2” and “D3” havethe analogous meanings for the B and D genomes. Mutants of each of thesenine possible types were identified among the collection of 34 mutants.

The extent of the chromosome deletion in each of the 34 mutants wasdetermined by microsatellite mapping. Microsatellite markers previouslymapped to the long arm of chromosomes 2A, 2B and 2D (Table 12) weretested on these mutants to determine the presence or absence of eachmarker in each mutant. Mutant plants in which either all or most of thespecific chromosome microsatellite markers were retained, based on theproduction of the appropriate amplification product in the reactions,were inferred to be relatively small deletion mutants. Such mutants werepreferred, considering that it was less likely that other, importantgenes were affected by the mutations. The identified mutants and theresults from the microsatellite mapping are summarized in Table 13.

Crossing of Mutants.

Mutant plants that were homozygous for smaller deletions as judged bythe microsatellite marker analysis were selected for crossing togenerate progeny plants and grain which had mutant SBEII alleles onmultiple genomes. F, 1 progeny plants from the crosses were selfed, andF2 seed obtained and analysed for their SBEII genotype. Screening 12such F2 populations led to the identification of 11 differentcombinations of mutant alleles (“double nulls”) (Table 14). The doublenull combination of the B1D1 genotype was not obtained in the twelfthcross in spite of screening more than 1200 F2 progeny of that particularcross. One possible explanation for this might be the presence of acritical gene in the vicinity of the SBEII locus in the B and D genomes,but not in the A genome, and hence the combination of the B1 and D1double null mutations might render the seed non viable. Twenty sevencombinations of double-null mutants are theoretically possible, and moreF2 populations will be screened to identify the other combinations.

Example 6: Amylose Content of Single and Double Null SBEII Mutants ofWheat

The percentage of amylose in the grain starch of single and double nullplants described in Example 5 was determined using the iodometric methodas described in Example 1. A scatter diagram plotting amylose content(Y-axis) against the mutant line number (X-axis) is shown in FIG. 4. Theamylose content in the mutant grains ranged from 27.3 to 38.7%. Theamylose content of wild-type (unmutagenised) Chara samples ranged from27.4% to 29.5%. Twenty six lines recorded an amylose content of above34%. It was observed that of these 26 lines, 20 were double nulls, ofwhich some were replicates from the same cross, of either Type 1 or Type2 combinations. In other words, there was a trend in significantlyincreasing amylose content in Type 1 and Type 2 double null combinationscompared to the amylose content in single null grains.

Importantly, and unexpectedly prior to this study, none of the doublenull mutant grains had starch with greater than 40% amylose. Thisincluded the A1B1, A1D1 and B1D1 genotypes which each contained fourSBEIIa and four SBEIIb null alleles and retained two wild-type SBEIIaand two wild-type SBEIIb alleles. This observation was consistent,however, with the prediction made from the data in Example 2. It wastherefore concluded that to obtain wheat grain with more than 40%amylose by combining mutations, the grain needed to have more than fourmutant alleles of SBEIIa, or alternatively, if only four mutant allelesof SBEIIa were present, more than four mutant SBEIIb alleles incombination with the four SBEIIa alleles, preferably all six SBEIIbgenes being mutant. It was also suggested from the data that the SBEIIagenes on each of the A, B and D genomes were expressed at similar levelsrelative to each other, i.e. SBEIIa expression in breadwheat was notpredominantly from any one genome.

It was interesting to note that the “A3” and “A3D3” genotypes had lowamylose contents consistent with the data in Example 2, confirming thatSBEIIb had a lesser role in determining amylose content in wheatrelative to SBEIIa.

Example 7: Crosses in Attempts to Create Triple Null Mutants

In order to create mutant lines with more than four SBEIIa mutantalleles, some of the single null and double null lines were crossed andthe F2 progeny of these crosses analysed using the diagnostic PCRassays. The assays tested for the presence of the three SBEIIa and threeSBEIIb genes and were therefore used in an attempt to identify plantswhich had null mutations in the SBEIIa and/or SBEIIb genes in each ofthe A, B and D genomes (triple null lines for SBEIIa and/or SBEIIb). Thecrosses that were carried out in a first experiment and the genotypes ofthe parental lines and potential triple null F2 progeny are listed inTable 15.

Starch granule morphology was analysed by microscopy of selected normallooking and shriveled/shrunken F2 seeds from these crosses. Sixshriveled/shrunken seeds were selected, 5 from the 08/dd cross and 1from the 08/bb cross, each of which were obtained from crosses between aD2 single null parent plant and an A1B2 double null parent plant. Eachof the six seeds showed severe distortion of starch granules, showingabnormal, distorted shapes for most granules in the seeds which wassimilar to granules observed in transgenic seeds with elevated amyloselevels (Example 2). Inspection of a number of shriveled/shrunken seedsand selected odd looking seeds from the other crosses revealed noaltered starch granule morphology, indicating that the phenotypeobserved in 08/dd and 08/bb seeds was genotype specific and not due todevelopmental problems during seed development.

Starch isolated from 6 of the seeds having distorted starch granules waspooled and tested for amylose content using the iodometric method asdescribed in Example 1. The amylose content of the pooled sample wasmeasured to be 67% (Table 16). Amylose levels in the wild-type seeds(control) of cultivars Cadoux and Chara were approximately 35%.

Genotypic Analysis of Seeds with Altered Starch Granule Morphology.

The seeds from the crosses 08/dd and 08/bb with altered starch granulemorphology were sown and the resultant plants grown in the greenhouse.DNA extracted from the plants was analysed using the genome specificprimers for SBEIIa and SBEIIb described in Example 3. Results from thePCR assays indicated that each of these seeds were homozygous doublenull mutants with an A1B2, B2D2 or A1D2 genotype while the third(wildtype) gene was present in either the homozygous or heterozygousstate. DNA from these plants were further tested using quantitative PCR(Real-time PCR, Rotorgene 6000) using genome specific individual primerpairs to assay the presence or absence and the homozygosity orheterozygosity of the 3 SBEIIa genes in the plants. The primer pairsused for SBEIIa were Snp6for/Arev5 (SEQ ID NO: 51/SEQ ID NO: 61) (Agenome, 205 bp amplification product), BSnp4/Arev5 (SEQ ID NO: 55/SEQ IDNO: 61) (B genome, 494 bp amplification product) and DSnp7for/Drev1 (SEQID NO: 58/SEQ ID NO: 62) (D genome, 278 bp amplification product). Inorder to normalize the SBEIIa amplification reactions, a primer pair(SJ156/SJ242) which amplified a 190 bp product from the CslF6 gene,which is a cell-wall biosynthesis gene expected to be equally expressedin all of the plants and located on wheat Chromosome 7, was used incontrol amplifications. DNA from a wild-type plant from the mutagenisedpopulation, designated 2B2, and from wild-type cv. Chinese Spring (CS)were used as control templates. The relative concentration valuesgenerated in the reactions with the SBEIIa primers were normalised withthe value for Cslf6 primers for each template DNA preparation. Thevalues for the potential triple null plants and CS were calculatedrelative to line 2B2.

Out of these three primer pairs, the D genome primers produced a clearsingle band for one plant designated as S14 which enabled quantitation.No bands were obtained for the SBEIIa genes on the A and B genomes ofS14, indicating it was homozygous for the mutant alleles on thesegenomes. The quantitation indicated that S14 had approximately 30-50% ofthe D allele complement compared to 2B2 whereas CS gave a value ofapproximately 95% of 2B2 for the D genome SBEIIa gene. This showed thatS14 which gave seed with amylose levels of about 67% was homozygous forSBEIIa null mutations for two of the genomes (A and B) and heterozygousfor the third genome (D), in addition to being homozygous for SBEIIbnull mutation in the A genome. That is, S14 had an A1 (homozygous), B2(homozygous), D2/+(heterozygous) genotype. In a similar fashion, thequantitative PCR showed that plant designated as S24 had a B2(homozygous), D2 (homozygous) and A1 (heterozygous) genotype, The PCRanalysis showed that the remaining 5 plants had the following genotypes:08dd9-B4 was homozygous for an A1B2 genotype i.e. homozygous mutant forSBEIIa and SBEIIb on the A genome, homozygous mutant SBEIIa andwild-type SBEIIb on the B genome and homozygous wildtype for both geneson the D genome, while 08bb11-D9 was homozygous for a B2D2 genotype andS28 and S22 were homozygous for an A1D2 genotype

Analysis of F3 Seeds.

Seeds of the S28, S22, S14 and S24 lines were sown in the greenhouse,the resultant plants were selfed, and seeds (F3 generation) obtainedfrom each plant. It was observed that the fertility of the plants wasaffected, in that the number of seeds per head and the percentage ofspikes which were fertile were significantly reduced compared to wildtype, single null and other double null mutants grown at the same timeand under the same conditions, but not abolished (Table 17).

Starch granule morphology was determined by light microscopy on 100-200seeds from each of the lines S28, S14 and S22. From the line S22, 102 F3seeds were identified with distorted starch granules from among 200seeds tested. The data revealed a distortion of the segregation ratiosaway from the expected 1:2:1 (homozygous mutant:heterozygote:wild-type)with a higher number of normal phenotypes than expected. In order to seewhether a homozygous plant with a high amylose phenotype could beidentified, 102 seeds with distorted granules were placed in conditionssuitable for germination. Sixty one out of the 102 seeds germinated. DNAfrom these 61 plants were analysed by SBEIIa genome specific PCR and all61 plants appeared to be double null of an A1D2 genotype, with nohomozygous triple nulls identified. The wild-type SBEIIa gene on the Bgenome was shown to be heterozygous i.e. both wildtype and mutantalleles were present for the B genome.

The 41 seeds which had distorted starch granules but had not germinatedwere analysed for their SBEIIa genotype. Many of these were observed tobe triple nulls, i.e. showing an absence of any amplification productfor the SBEIIa genes and therefore having six null alleles for SBEIIa.This confirmed that the triple null seeds could be generated but theseseeds had defects that affected germination. Embryos from some of theseseeds were excised and cultured using tissue culture media underconditions to promote germination of the embryos. Some embryosgerminated successfully, resulting in green plantlets. However, whenthese plantlets were transferred to soil, they grew poorly and did notproduce fertile wheat plants.

From these data, it was concluded that a homozygous triple null mutantseed based on the HIB-generated deletion mutations, and plantletsderived from these seed and having six null SBEIIa alleles and entirelylacking SBEIIa, were recoverable from these crosses, but were affectedin germination and growth, indicating an essential role for some SBEIIain these processes. In contrast, the double null mutants for SBEIIawhich were heterozygous for the third null allele and therefore havingfive null SBEIIa alleles were recovered, grew normally and were fertile,albeit with reduced fertility.

Protein Expression Analysis of Line S28.

SBEIIa protein expression in developing endosperms obtained from onewhole spike from an S28 plant was analysed by Western blotting using aSBEIIa specific antibody. All 15 endosperms in the spike showed apattern lacking both A and D genome isoforms of SBEIIa (AD double null)with only one SBEIIa band present, expressed from the B genome. Out ofthe 15 endosperms, 7 had a B genome SBEIIa expression level considerablylower than the others and that of the control line, NB1. Based on theband intensity, the SBEIIa expression in each endosperm was quantitated.

The remaining starch granules from the endosperms were purified using90% Percoll. Following resuspension in 200 μl water, the granules wereexamined microscopically. It was observed that all endosperms having anexpression level of SBEIIa which was less than about 36% of thewild-type had starch granules with distorted morphology typical of ahigh amylose phenotype. A range of SBEIIa protein expression levels wereobserved in the developing grains from one spike from an S24 plant, downto less than 5% of wild-type. Endosperms with the lower levels of SBEIIaalso showed altered starch granule morphology; the phenotypes weretherefore completely correlated in this experiment. SBEIIb expressionlevels in all these endosperms were also analysed using a SBEIIbspecific antibody. The results clearly showed that there was aconcomitant reduction in the SBEIIb expression corresponding to thereduction in SBEIIa expression.

Discussion.

The analysis of the seed from plants with the A1B2 mutant genotype(summarised in Table 18) having four mutant SBEIIa alleles indicatedthat the amylose content was elevated only slightly for that genotype,yielding an amylose level of less than 40%. In comparison, the data fromthe S14, S22, S24 and S28 seeds demonstrated that the addition of thefifth SBEIIa mutant allele elevated the amylose level to about 67%.Accordingly, the increase in number from four SBEIIa null alleles to aminimum of five mutant SBEIIa alleles was critical to increasing theamylose level to greater than 50% (w/w), indeed greater than 60% (w/w).This conclusion fitted with the predictions made from the data inExample 2. The observed relationship of the allelic composition to theamylose content indicated that the total number of SBEIIa mutant allelesin the plant was important in determining the amylose content (Table18). It was also concluded that the number of SBEIIb mutant alleles alsoplayed a role, although less important than the number of SBEIIa mutantalleles.

It was also concluded that homozygous triple null mutant seeds andplantlets having six null SBEIIa alleles and entirely lacking SBEIIacould be generated from the single null mutants containing HIB-generateddeletions, but these were affected in germination and growth, indicatingan essential role for some SBEIIa in these processes. In contrast, thedouble null mutants for SBEIIa which were heterozygous for the thirdnull allele and therefore having five null SBEIIa alleles wererecovered, grew normally and were fertile.

Example 8: Further Attempts to Produce Triple-Null Mutants EntirelyLacking SBEIIa or SBEIIb

The observed inability to generate a triple null mutant completelylacking SBEIIa in the Example above may have been dependent on theparticular mutant plants used as parents in the crossing. To test this,a second set of crosses using additional parental mutants, also obtainedfrom the HIB-mutagenesis, was carried out, summarised in Table 19. TheF2 seeds from 38 crosses were harvested and DNA extracted. At least 96DNA each from 25 crosses, 12 of which are from crosses aimed atproducing an A1B2D2 genotype (triple null mutant) but using differentparental lines than described in Example 7, was screened by PCR todetermine the trend of segregation. No viable triple nulls wereidentified from any of these crosses. Recovery of the double nulls alsovaried depending on the cross, but in most cases the expected genotypeswere obtained. F2 seeds from six of the A1B2D2 crosses were alsoscreened microscopically to identify seeds having a high amylosephenotype. Such seeds were identified at a moderate frequency.

Screening of Seeds from the A2B2D2 Cross, 08/Mm-1.

Among the crosses listed in Table 19, 12 were crosses between a parentwith an A2 genotype and a parent with a B2D2 genotype, i.e. both parentswere wild-type for all three SBEIIb genes, with the aim of generatingtriple null SBEIIa mutants having the A2B2D2 genotype. DNA preparationsfrom approximately 672 F2 seeds obtained from the 08/mm-1 cross werescreened by PCR. Segregation ratios were distorted from the expectedMendelian ratios, with significantly fewer double nulls identified thanexpected (Table 20). Nevertheless, all possible combinations of doublenull mutations were identified in viable seed. No triple nulls of theA2B2D2 genotype were identified amongst the 672 seeds, even though byMendelian segregation about 10 would have been expected.

In parallel, F2 seeds of the 08/mm-1 cross were screened by microscopyto identify any seeds with a high amylose/distorted starch granule (HA)phenotype. Of 576 F2 seeds that were screened, no seeds were identifiedwith the HA phenotype. This population of seeds should have included alow frequency of seeds having 5 mutant SBEIIa alleles, being homozygousmutant in two genomes and heterozygous mutant/wild-type in the thirdgenome for SBEIIa. The observed lack of seeds with a HA phenotype in theA2B2D2 cross indicated that 5 mutant SBEIIa alleles, in the absence ofany SBEIIb mutant alleles, did not appear to be sufficient to provide ahigh amylose (>50% amylose) phenotype. That is, a reduction in SBEIIblevels relative to wild-type in addition to the greatly reduced SBEIIalevel in the context of 5 mutant SBEIIa alleles and one wild-type SBEIIaallele, or an equivalent level of SBEIIa activity in an endosperm havingpartial loss of function mutations in one or more SBEIIa genes, wasneeded to provide greater than 50% amylose.

Screening of F2 seeds from eleven additional crosses between singleSBEIIa mutant parents (wild-type for SBEIIb) and double SBEIIa mutantparents on the other two genomes also did not identify any viable triplenull mutant seed of the A2B2D2 genotype.

Crosses Involving Type 3 Mutations.

Crosses involving Type 3 mutations were carried out with the aim offinding homozygous mutants having two, four or six mutant SBEIIb allelescombined with four mutant SBEIIa alleles, and determining the phenotypeof the resultant plants and its grain. Table 21 summarises the resultsof screening of crosses involving Type 3 mutations. Triple nulls wereidentified from A3B3D3 and A3B2D2 crosses, both of which showed wildtype starch granule morphology.

Example 9: Further Screening for High Amylose Mutants

In further attempts to produce triple null SBEIIa mutants fromidentified single mutants, an altered strategy was adopted. Thisstrategy added the step of some initial backcrosses of the singlemutants after their identification, in order to remove unlinked andunrelated mutations from the M2 plants having the single SBEIIamutations. This was included to reduce the effect of the mutatedbackground, due to the high level of mutagenic treatment used, whichwould have produced additional mutations in the plants independent ofthe desired SBEIIa mutations that could have detrimental effects whenthe mutations were combined. These initial backcrosses were carried outby crossing the M2 mutants with plants of either winter wheat cultivarApache or spring wheat cultivar Chara.

Initially, 13 crosses were performed to combine mutations on all threegenomes, and molecular analysis was done on DNA from 21,400 F2 halfseeds, with the second half of each seed retained to preserve the line.A preliminary screening to detect mutations used dominant SSR markerswhich were genome specific for SBEIIa or SBEIIb. From this, 21 seedswere identified as being putative triple null mutants and 793 seeds asputative double mutants (Table 22) by the absence of genome specificamplification products.

Q-PCR TaqMan-Based Assays of Wheat Seed Genotypes.

The first round of screening using dominant markers as described abovecould not distinguish between seeds that were heterozygous or homozygouswild-type for any one SBEIIa gene. A TaqMan-based PCR assay wastherefore developed to distinguish heterozygotes and homozygotes for theSBEIIa gene on the third genome, and to confirm the genotypes from theinitial screening. Because the TaqMan analysis was done on half seedsand because wheat endosperm is triploid (3n) for each genome, two typesof profiles were possible for heterozygous endosperm for the wild-typeSBEIIa allele on the third genome, either 2n, where the wild-type allelewas provided by the maternal parent, or 1n, where the wild-type allelewas provided by the paternal parent through the pollen. Q-PCRTaqMan-based Assays used the Applied Biosystems 7900HT Fast Real TimePCR System (ABI, Foster City, Calif.) to detect the copy number of theSBEIIa gene on the third genome of putative double mutant wheat seeds.The assays used genomic DNA extracted from half seeds by magnetic beadmethods (Nucleomag, Cat Ref No. 744 400.24). DNA was loaded on 384-wellplates and duplex Q-PCR reactions were performed in duplicate for eachplate. The PCR reactions were designed to amplify a 65 bp fragment fromexon 21 of the SBEIIa genes using the primers SBE2a QPCRABDF4 (forwardprimer): 5′-ACGATGCA CTCTTTGGTGGAT-3′ (SEQ ID NO: 31) and SBE2aQPCRABDR4 (reverse primer): 5′-ACTTACGGTTGTGAAGTAGTCGACAT (SEQ ID NO:32). The probe used to deliver the fluorescent signal during Q-PCRreactions was SBE2a QPCRABDS4 (TaqMan® probe MGB, FAM)5′-CAGCAGGCTTGATCAT-3′ (SEQ ID NO: 33). A sequence from an endogenousgene, GamyB, was used as an internal control to normalize the signalvalue of each sample, using the primers GamyB1F (primer forward):5′-GATCCGAATAGCTGGCTCAAGTAT-3′ (SEQ ID NO: 34) and GamyB2R (primerreverse): 5′-GGAGACTGCAGGTAGGGATCAAC-3′ (SEQ ID NO: 35). Reactionconditions were as follows: “hot start” (95° C., 10 min) followed by 40cycles of denaturation (95° C., 15 sec), annealing (58° C., 60 sec).Reaction products were analysed using Relative Quantification managersoftware integrated to the 7900HT Fast Real Time PCR System.

Using this TaqMan assay, all of the 21 putative triple null mutants wereconfirmed to be double nulls, not triple nulls. The incorrectidentification in the initial screening was thought to be due to falsenegatives, perhaps caused by poor template DNA quality. When 14 of theseeds were examined for starch granule morphology by light microscopy,all 14 were observed to have a wild-type granule phenotype, which wasconsistent with the seeds being double null mutants, not triple nullmutants. The assays also identified a few putative double mutant seedsthat were 2n heterozygous on the third genome, from crosses M76, M77,M82, M83 and M86. However, those results need to be confirmed as it wasdifficult to distinguish the 2n heterozygous genotype from the 3nhomozygous genotype, even in the presence of the double null SBEIIabackground. This will be confirmed in the next generation of progeny.The assays also showed that no SBEIIa double null mutants that wereheterozygous mutant SBEIIa/wild-type on the third genome were obtainedfrom crosses M79, M81, M74, M75, M78 and M80. The crosses M84 and M85gave the highest number of clearly identified homozygous double nullSBEIIa mutants which were good candidates for being 1n heterozygotes(mutant SBEIIa/wild-type) on the third genome. Some 2n heterozygoteswere also identified but need to be confirmed.

In these crosses, the numbers of single and double null SBEIIa mutantswas lower than the frequency expected from Mendelian segregation. Thisdistortion of segregation was further studied. Where the expectedfrequency of homozygous single mutants should have been 25%, in somecrosses the frequency was much lower, ranging from 1% to 25%. The numberof double homozygous mutants in the progeny of crosses to produce triplenull mutants should theoretically be around 6% (¼*¼) per combination (6%AB, 6% AD, and 6% AB). The actual number of double mutants identifiedwas much lower and ranged from 0 to 5.2%. This suggested that somecombinations of mutations were detrimental to the plant, for example toseed development, leading to a lower recovery of combinations ofmutations than expected. Two crosses, M74 and M75, gave the lowestfrequencies compared to the expected. It was noted that the parents usedin those crosses had not been backcrossed with Apache or Chara beforethe crosses were performed, suggesting that additional, unrelatedmutations in the parents arising from the mutagenic treatment may havehad a role in the distortion of segregation ratios. Even for crosses M76and M86 which gave a higher number of single mutants, the frequency ofdouble null mutants was low, in particular for some combinations. Forexample, for cross M76 the frequencies of single nulls in the D genomeand the A genome were 23% and 17%, respectively, while the frequency ofthe double null mutants in both the A and D genomes was only 0.8%. Thissuggested that some combinations of SBEIIa mutations were less favorableto the plant than others, and consequently counter-selected. Theproportion of mutants containing five mutant SBEIIa alleles (doublenulls which were heterozygous mutant on the third genome) was also verylow. The expected frequency would be 9% (¼*¼*½*3) while the highestobserved percentage was 1.1% for the M84 and M85 crosses.

Correlation between frequencies of homozygous single and double mutantsin M74 and M75 crosses was quite good for SBEIIa mutations on the A andD genomes (0.789 and 0.558 respectively) while much lower (0.386) forthe B genome. A possible explanation would be that one of the parents(19.832 (D1)/20.257(A2) [08/b12]) used in M74 and M75 crosses was aheterozygote in the first place rather than a double homozygous mutant.

Under these conditions, the probability of obtaining a triple nullmutant (6 null mutant SBEIIa alleles) was very low and much less thanthe expected frequency of 1/64. However, selfing of the double mutantswhich were also heterozygous on the third genome, in particular from theM84 and M85 crosses, is expected to confirm whether the triple nullmutants are recoverable from these parental mutants. The progeny of theselfed plants will be analyzed to identify any triple mutant seed.

Example 10: Screening for Mutant Wheat Seeds by NIR

A rapid, non-destructive and high throughput method was developed toscreen single seeds for a phenotype that was associated with highamylose content. The PCR-based screening methods described in Examples4-6, while successful in detecting mutants in a population of 15,000seeds, required DNA preparation from each half seed after cutting eachseed manually, and so was time-consuming and tedious. It was determinedthat Near Infrared Spectroscopy (NIRS) could be used to distinguishbetween the high amylose and normal amylose phenotypes. Near InfraredRed Spectroscopy (NIRS) is a non destructive technology that has beenused to determine some wheat seed properties (McClure, 2003). Wheatsingle seed NIRS analysis for a waxy starch phenotype (low amylose) hasbeen developed on durum wheat by Delwiche et al. (2006). Dowell et al(2009) developed an automated single seed NIR sorting system to separatewaxy, partial waxy and normal durum and hexaploid wheat. To ourknowledge, this method has not been used previously to distinguish highamylose seeds in hexaploid wheat.

Development and Validation of Scaled Down Biochemical Reference Methodto Measure Apparent Amylose Content in Ground Seed Material.

In order to calibrate NIRS measurements according to apparent amylosecontent in individual seeds, a mathematical model had to be establishedto correlate NIRS spectrum data and a biochemical method measuringapparent amylose content on the same sample, in this case single seeds.Standard iodometric methods, for example, the method described inExample 1, routinely use a quantity of seeds which are combined beforestarch solubilisation, providing bulked (combined) starch which isnormally defatted prior to colorimetric measurement of the amylosecontent based on iodine binding. To be suitable for use for NIRScalibration purposes, this method was modified, simplified and scaleddown to allow measurement of apparent amylose content in single seeds,thereby to allow for variation in amylose content between seeds. Theterm “apparent amylose content” is used in this context because themodified method did not purify the starch from the ground grain, thelipids interacting with the amylose in the starch were not removed, andthe results were expressed as percentage of fresh seed weight ratherthan as a percentage of the isolated starch from the seed. For thesereasons, the values obtained for “apparent amylose content” were muchlower than the values obtained using the standard method as described inExample 1.

As a first step, this method was developed by assessing the linearitybetween the colorimetric response and amylose content using ground wheatgrain without starch purification. The high amylose material used forthis was wheat grain transformed with the hp5′-SBEIIa construct andhaving reduced SBEIIa (WM, Line 85.2c, see Example 2) and wheat with thenormal amylose level which was a wild-type wheat (WMC) grown at the sametime and under the same conditions. Ground WM grain contained about 80%amylose as determined by the standard method of Example 1, while groundWMC grain had an amylose content of about 25%. Samples with differentratios of WM to WMC were prepared from ground seed material but notfurther purified. Approximately 17 mg samples were used for the assay.The WM and WMC mixtures were weighed accurately into 1.5 mlmicrocentrifuge tubes. To solubilise the starch in the samples, 1 ml ofDMSO was added per 17 mg of sample and then the mixtures heated in a 95°C. water bath for 90 min with occasional vortexing. A 10 μl aliquot fromeach mixture was added to 1.98 ml of water and treated with 10 μl 0.3%12+3% KI in 0.01N NaOH solution. The absorbance of each mixture wasmeasured at 605 nm and absorbance values were converted to percentamylose using a standard curve. The standard curve was made using maizeamylopectin (Sigma catalogue No. A7780) and potato amylose (Sigma,A0512) in ratios from 0% to 100% amylose and treated the same way as theground wheat samples.

The results showed a linear relationship between the level of WMincorporation and the apparent amylose content, showing that thesimplified iodometric method could be used for NIRS calibration and thatstarch purification was not needed for this purpose.

Testing the Biochemical Reference Method to Measure Apparent AmyloseContent in Half Seeds.

Seeds from the WM and WMC (control) lines obtained from field trialexperiments conducted in Arizona and Washington were used for thistesting. In total, 47 half seeds with embryos removed were individuallyplaced in 1.5 ml microcentrifuge tubes and weighed accurately beforeaddition of 0.6 ml of DMSO to each. The tubes were incubated in awaterbath at 95° C. for 20 min after which the samples were crushed inthe tubes using a glass rod. The volume of each mixture was adjusted toprecisely 1 ml of DMSO per 17 mg of sample after which the tubes wereincubated at 95° C. in a waterbath for another 70 min with occasionalvortexing. Apparent amylose was measured by taking 10 μl aliquots ofeach mixture and treating them with 10 μl 0.3% 12+3% KI in 0.01N NaOHsolution and diluted to 2 ml with H₂O, as before. Absorbance of eachsample was measured at 605 nm and absorbance values were converted topercent “apparent amylose” using a standard curve as described above.

Using this method, the apparent amylose content of WM seeds ranged from20% to 41% (average 27%) while the apparent amylose content of WMC seedsranged from 7.5% to 17% (average 11.4%). The reasons why these valueswere much lower than the amylose content as determined by the method ofExample 1 are described above. This simplified method therefore allowedseeds with high amylose to be distinguished from those with wild-typeamylose content.

NIRS Calibration.

Single seed NIRS scans on WM and WMC seeds were obtained using a MultiPurpose Analyser (MPA) NIRS spectrometer (Bruker Optics, F-77420 ChampsSur Marnes, France). Each seed was placed at the bottom of a glass tubewrapped with aluminium foil and scanned twice. Spectra were recordedusing a Bruker MPA Multi-Purpose-Analyser spectrometer (Bruker Optics)fitted with a fiber probe. Spectra were recorded using 32 scansreference and 16 sample scans over the range 4000-12,500 cm⁻¹ at aresolution of 16 cm⁻¹ resulting in 1100 data points. The fiber opticprobe used was the IN 261 probe for solids.

To determine the correlation between apparent amylose levels and NIRreadings, 226 individual WM or WMC seeds with apparent amylose contentsranging from 6 to 44% were analysed. First, duplicate NIRS spectra wereacquired for each seed, after which the apparent amylose content wasbiochemically measured for each seed according to the method describedabove. Spectral outliers (6 samples) were identified as spectra thatwere abnormal compared to the spectra of the entire data set andeliminated, and the remaining spectra analysed with NormalisationMin-max pre-treatment. The Partial Least Square software with full (oneout) cross validation was used to create the model. The spectral windowused for the model development was 9827-7150 cm⁻¹ and 6271-4481 cm⁻¹.The number of PLS factors used to develop the calibration was 14. Theaccuracy of the calibration model was expressed by the standard error ofcross validation (SECV) and the coefficient of determination (R²). Theefficiency of a calibration was shown by the RPD which is the ration ofthe standard error of prediction (RMSECV) to the standard deviation ofthe reference data of the set.

A positive correlation (R²=0.702) was obtained between the biochemicaldata and the NIR spectral data (FIG. 15). It was concluded that themodel was robust enough to distinguish high amylose wheat seeds fromnormal amylose wheat seeds, but not yet accurate enough to preciselymeasure the amylose content in any one seed. The method was thereforecapable of screening a very large population of seeds to enrich forgrains with high amylose phenotype. This was validated as follows.

NIRS validation.

To validate the NIR method in distinguishing high amylose grain andcontrol grain, 60 more WM seeds and 34 WMC seeds were scanned twice byNIR and the predicted apparent amylose contents calculated. When theapparent amylose values so determined were plotted to obtain thedistribution profile for the WM and WMC populations, it was seen thatthe two groups were mostly separated with a slight overlap (FIG. 16).According to these results, seeds having a predicted apparent amylosephenotype determined by NIRS equal to or above 30% could be consideredas good candidates to be high amylose seed.

NIRS Screening of F2 Seeds from Wheat Crosses.

NIRS screening was carried out to detect mutant seeds having highamylose content. The screening used 2,700 F2 seeds from two differentcrosses: M80 and M85 which were, respectively: 21.142(B2)/TypeI-20257(A1) [08/h-111]//Type I-19.832 (D1)/CHARA and 5.706 (D2)/21.668(B2)//20.257 (A1)/CHARA. The screening was therefore aimed atidentifying seeds with an A1B2D1 or A1B2D2 genotype, respectively. TwoNIRS spectra were recorded per seed as described above

Seeds which gave a predicted apparent amylose value above 34% in atleast one of the two duplicate screenings were first selected forfurther analysis. Out of the 2,700 seeds, 27 seeds were selected andwere next assessed by light microscopy to determine the starch granulemorphology. Each seed was carefully scraped to preserve the embryo, yetobtain enough endosperm material to be examined. Four seeds of the 27were observed to have distorted starch granule morphology. These fourseeds happened to have had the highest predicted apparent amylosecontent from the NIR screening and were the only ones where bothpredicted apparent amylose values were above 30%. The other 23 seedsshowed normal (wild-type) granule morphology.

Molecular Data on Seeds Selected by NIRS Screening.

PCR analysis was carried out on the four seeds to determine the SBEIIagenotype of each. Initial assays used dominant PCR markers which showedthe presence or absence of each SBEIIa gene on the three genomes. Threeof the seeds were shown to be double null mutants while the fourth was aputative triple null mutant. However, when tested further with aco-dominant PCR marker (see below), all of the four seeds were shown tobe double null mutants for SBEIIa (i.e. lacking SBEIIa in two genomes)and heterozygous for a mutant SBEIIa gene on the third genome.Therefore, these seeds contained 5 mutant SBEIIa alleles and at leasttwo mutant SBEIIb alleles.

When the embryo from each seed was placed under conditions to germinate,none of them germinated successfully, perhaps because they were toodamaged or the combination of mutations was too detrimental.

In order to try to identify more candidates, further NIRS screening wasperformed on more F2 progeny seeds from the M80 and M85 crosses, withless stringent selection of candidate seeds. The selection criterion forthe second screen was that one of the predicted apparent amylose valueshad to be above 30% and the second one at least 23%. A new set of 22seeds was selected for starch granule evaluation by light microscopy.Out of those 22 candidates, 1 seed, BD85;9F08 (P279-F08-834), showed adistorted starch granule phenotype. This mutant was further analysed byPCR and shown to be a double null SBEIIa mutant on the A and B genomesand heterozygous for the mutant SBEIIa gene on the D genome. It wassuccessfully germinated for multiplication.

Example 11: Detection of Alleles of Starch Branching Enzyme with AlteredStarch Binding Affinity

Populations of mutagenised wheat grains, produced by treatment with thechemical mutagens sodium azide or EMS were screened to identify mutantswhich had point mutations in SBEIIa genes and therefore potentiallyreduced, but not abolished, SBEIIa-A, -B or -D activity, or SBEIIb-A, -Bor -D activity (partial mutants) relative to wild-type wheat. Screeningfor mutants was based on measuring the amount of the SBEIIa or SBEIIbproteins by using Western blotting with antibodies specific for SBEIIaor SBEIIb (see Example 2), or by affinity-based techniques, as follows.This screening was also expected to detect mutants with point mutationswhich lacked SBEIIa-A, -B, or -D activity entirely as well as themutants with partial activity.

Native gel electrophoresis of protein extracts from grain includingstarch branching enzymes through a polyacrylamide matrix containingglycogen, amylopectin, amylose or β-limit dextrin (affinity gelelectrophoresis) provides a method for identifying alleles of SBEIIa orSBEIIb which encode SBEIIa or SBEIIb with altered starch bindingcapacity. Given that the active site of starch branching enzymescontains a starch binding site, SBEII polypeptides with altered bindingefficiency are likely to have alterations in catalytic rate and/oraffinity. In particular, polypeptides with reduced binding efficiencywere expected to have reduced SBEII activity.

The following methods were used, based on Morell et al., (1997); andKosar-Hashemi et al., (2006) with some modifications.

Preparation of Proteins.

Soluble proteins were extracted by homogenising the isolated endospermsfrom developing seeds (about 15 days post-anthesis) in 50 mM phosphatebuffer, pH 7.5 containing 5 mM EDTA, 5 mM DTT, 0.4% protease inhibitorcocktail and 20% glycerol. After centrifugation at 14,000 g for 10 minthe supernatant was used for the gel electrophoresis. Proteinconcentration in the extracts was estimated using a

Coomassie Plus Protein Assay Reagent.

Affinity Electrophoresis.

In a two-dimensional (2D) affinity electrophoresis technique forseparating SBEIIa protein isoforms, aliquots (40 or 100 μg) of theprotein extracts were loaded onto the first dimension gel, anon-denaturing polyacrylamide gel cast in a Hoefer SE600 vertical 16 cmslab gel unit. The resolving component of the second dimension gel was a6% non-denaturing gel (14×16 cm or 16×16 cm, 1.5 mm thickness)containing 10% glycerol with an appropriate amount of polysaccharidetarget (amylopectin, β-limit dextrin or glycogen) immobilised within thegel structure. A stacking gel (polysaccharide-free) was poured to 1 cmfrom the top of glass plates forming using a comb to form wells. Gelswere run overnight at 4° C. at constant voltage (100V for glycogen andβ-limit dextrin and 135V for amylopectin containing gels).

Alternatively, a one dimensional system was used to separate SBEIIaproteins in which protein extracts (20 μg) were loaded onto anon-denaturing polyacrylamide gel. The resolving component of the gelwas a 6% non-denaturing gel containing 10% glycerol with 0.15% ofβ-limit dextrin immobilised within the gel structure, while the stackinggel was polysaccharide-free. Gels were run at 4° C. at constant currentof 20 mA per gel and maximum voltage of 200V.

SBEIIb proteins can also be separated on a Bis-Tris 4-12% gradient gel(Invitrogen). The gel is run at 4° C. at constant current of 20 mA pergel and maximum voltage of 200V.

Immunological Detection.

For immunochemical detection of the SBEII proteins followingelectrophoresis, the proteins were transferred from the gels tonitrocellulose membranes using a TE 70 PWR semi-dry transfer unit(Amersham Biosciences). The transfer buffer contained 39 mM glycine, 48mM Tris, 0.0375% SDS and 20% methanol. Transfer was carried out for1-1.5 h with a constant current of 0.8 mA/cm². The membrane was blockedwith 5% skim milk prior to Western blotting using primary rabbitpolyclonal antibody specific for wheat SBEIIa.

The migration patterns of the SBEII isoforms encoded by the homeoallelesfrom the wheat A, B and D genomes showed differences between differentwheat varieties when analysed by the one-dimensional affinity gelelectrophoresis method. In some varieties, clear separation of the A, Band D homeoforms was possible, allowing the simple scoring ofpolymorphisms in mutagenised populations from those varieties. Forexample, affinity gel electrophoresis of protein extracts fromendosperms of the wild-type wheat varieties Sunstate and NB1 showed aclear separation of the SBEIIa-A, -B and -D isoforms. Branching enzymealleles with a reduced affinity for starch migrated a greater distancethrough the polysaccharide-containing polyacrylamide gel than therespective native homeoalleles. Lines containing alleles with reducedexpression or an absence of expression of a particular homeoallele wereidentified by presence/absence of a band in homozygous state and throughdensitometry to measure band intensity in heterozygous lines. Tovalidate this method, SBEIIa- and SBEIIb-mutant plants which wereidentified by genotypic analysis (Example 6) were confirmed to belacking specific SBEIIa or SBEIIb proteins by affinity gelelectrophoresis, consistent with their genotypes. These experimentsvalidated this protein analysis method for detection of mutants having areduction in amount or activity of an SBEII isoform.

Screening of a population of 2100 mutagenised wheat lines of the varietySunstate, treated with sodium azide as described in Zwar and Chandler(1995), using β-limit dextrin affinity gel electrophoresis led to theidentification of 18 mutants which had either altered mobility on theaffinity gels of one of the SBEIIa proteins (affinity mutants) or nullmutants for one of the SBEIIa genes based on a lack of detectableprotein encoded by that gene. The dissociation constant (Kd) ofstarch-enzyme interactions for each of the SBEIIa isoforms in one of theaffinity mutants was calculated by measuring the change in enzymemobility as a function of the β-limit dextrin concentration in a 1-Daffinity gel as described in Kosar-Hashemi et al., 2006. This affinitymutant had SBEIIa proteins with the following Kd values: 0.53 g/L, 0.52g/L and 1.69 g/L for the SBEIIa-A, SBEIIa-B and SBEIIa-D isoformsrespectively (FIG. 13). The higher observed Kd value for the D isoformcompared to that of the A and B isoforms indicated a lower, reducedaffinity of this isoform for binding to starch, indicating that thisline was an affinity mutant for the SBEIIa-D gene. The D-genome isoform(SBEIIa-D) of this line is expected to have a lower enzyme activity, butnot total loss of activity, compared to the other two isoforms. Thisexpectation is confirmed by SBEII activity assays in the presence ofnull alleles of SBEIIa-A and SBEIIa-B. The SBEIIa single mutantsidentified from the sodium azide mutagenised Sunstate population werethen crossed with the previously identified HIB double null mutants forisolating triple mutants that lack SBEIIa activity from two genomes withtotal or partial loss of activity from the third genome. Four crosses toisolate A1B2D2, two crosses each to isolate A2B2D2 and A2B2D1 and onecross to isolate A1B2D1 genotypes were performed. Examination of starchgranule morphology of F2 seeds from one of the A1B2D2 crosses bymicroscopy identified seeds with severely distorted starch granulessimilar to that is found in high amylose starches (at least 70%amylose). The genotype and amylose phenotype of these seeds is confirmedby analysing the SBEIIa alleles in the seeds and progeny and byextracting and analysing starch from the progeny grain. Eight crosseswere also performed between affinity single mutants to produce affinitydouble mutants of SBEIIa. This included crosses generated with the aimof isolating A2B2, A2D2 and B2D2 double affinity mutants. F2 progeny areanalysed by the methods described above to identify the doublehomozygous affinity mutants.

Example 12: Properties of Starch Granules and Starch from High AmyloseWheat Grain

Changes in Starch Granule Morphology and Birefringence.

Starch and starch granule properties were examined in the transgenichigh amylose wheat described in Example 2. Scanning electron microscopywas used to identify gross changes in starch granule size and structure.Compared to the untransformed control, starch granules from endospermshaving reduced SBEIIa expression displayed significant morphologicalalterations. They were highly irregular in shape and many of the Agranules (>10 μm diameter) appeared to be sickle shaped. In contrast,both A and B (<10 μm diameter) starch granules from endosperms havingreduced SBEIIb expression and unaltered SBEIIa expression were smoothsurfaced, spherical or ellipsoid in shape and indistinguishable fromwild-type wheat starch granules.

When observed microscopically under polarised light, wild-type starchgranules typically show a strong birefringence pattern. However, thebirefringence was greatly reduced for granules containing high amylosestarch. Less than 10% of the starch granules from lines having reducedSBEIIa expression and 70%-80% amylose content were birefringent whenvisualized under polarized light. For lines having essentially no SBEIIbexpression but with wild-type SBEIIa expression, no change inbirefringence was observed compared to non-transformed controls. In bothwild-type and SBEIIb-suppressed lines, approximately 94% of the starchgranules exhibited full birefringence. The data is given in Table 23.Loss of birefringence therefore correlated closely with high amylosecontent.

Amylose content of transgenic wheat grain. The amylose content oftransgenic wheat grain was assayed by two independent methods, namely aniodometric method and a size exclusion chromatography (SEC) method. Theiodometric determination of amylose content was based on measuring thecolour change induced when iodine bound to linear regions of α-1,4glucan, with reference to a standard curve generated using knownconcentrations of purified potato amylose and amylopectin, as describedin Example 1. The size exclusion chromatography method was based on theseparation, by column chromatography, of amylose and amylopectin whichhad not been debranched, followed by measurement of the starchconcentration in the fractions eluted from the column. Three genotypesof grain were analysed. Firstly, plants transformed with the hp-SBEIIaconstruct and having very low levels of SBEIIa expression; secondly,plants containing the hp-SBEIIb construct and having no detectableexpression of SBEIIb but wild-type for SBEIIa; and thirdly, thenon-transformed wild-type control (NB1). Grain from the plants lackingSBEIIb expression (008) had an amylose content of 27.3% determined bythe iodometric method and 32% by the SEC method. This was notsignificantly different to the amylose content of grain fromnon-transformed control line NB1 (31.8% iodometric, 25.5% SEC). However,in grain having the reduced SBEIIa expression (line 087) the amylosecontent was significantly elevated (88.5% iodometric, 74.4% SEC). Thedifference in these two figures for line 087 was thought to be thepresence of some “intermediate material” which binds iodine much likeamylose and was measured in the iodometric assay as amylose but wasseparated in the column chromatography with the larger amylopectin.

Chain Length Distribution of Starch by FACE.

Chain length distribution of isoamylase de-branched starch wasdetermined by fluorophore assisted carbohydrate electrophoresis (FACE).This technique provides a high resolution analysis of the distributionof chain lengths in the range from DP 1 to 50. From the molar differenceplot in which the normalized chain length distribution of thenon-transformed control was subtracted from the normalized distributionof the transgenic lines, it was observed that there was a markeddecrease in the proportion of chain lengths of DP 6-12 and acorresponding increase in the chain lengths greater than DP12 in starchfrom grain having reduced SBEIIa expression. No statisticallysignificant alteration in the chain length distribution of starch fromhp-SBEIIb lines was observed when compared to wild-type.

Molecular Weight of Amylopectin and Amylose.

Molecular weight distribution of starch was determined by sizeexclusion-HPLC (SE-HPLC). The HPLC system comprised of a GBC pump (LC1150, GBC Instruments, Vic, Australia) equipped with Auto Sampler (GBC,LC1610) and Evaporative Light Scattering Detector (ELSD) (ALLTech,Deerfield, USA). The Ultrahydrogel™ 1000 column, Ultrahydrogel™ 250column and guard column (7.8 mm×300 mm, Waters, Japan) were used andmaintained at 35° C. during HPLC operation. Ammonium acetate buffer(0.05 M; pH 5.2) was used as the mobile phase at a flow rate of 0.8 mLmin⁻¹.

The molecular weight of amylopectin in the starch of the reduced SBEIIagrain appeared to be much lower than that of amylopectin in the starchesof NB1 (wild-type, non-transgenic) and the reduced SBEIIb grain (peakposition of 7166 kDa versus 45523, 43646 kDa). In contrast, themolecular weight of amylose from the reduced SBEIIb grain was notsignificantly different compared to that of wild-type grain fromnon-transformed variety NB1. The data is in Table 24.

Total Starch Content in Endosperm of Wheat with Reduced SBEIIaExpression.

Analysis of total starch content in grain as a percentage of grainweight revealed a slight reduction in the endosperm starch content ofthe hp-SBEIIa line (43.4%) compared to 52% in the control and 50.3% inhp-SBEIIb line (Table 23). This indicated that there was some reductionin total starch synthesis when SBEIIa expression was reduced by theinhibitory construct.

Starch Swelling Power (SSP).

Starch swelling power of gelatinized starch was determined following thesmall scale test of Konik-Rose et al., (2001) which measured the uptakeof water during gelatinization of starch. The estimated value of SSP wassignificantly lower for starch from the reduced SBEIIa line with afigure of 3.51 compared to starch from the control (9.31) and reducedSBEIIb grain (10.74) (Table 23).

Starch Pasting Properties.

Starch paste viscosity parameters were determined using a Rapid ViscoAnalyzer (RVA) essentially as described in Regina et al., (2004). Thetemperature profile for the RVA comprised the following stages: hold at60° C. for 2 min, heat to 95° C. over 6 min, hold at 95° C. for 4 min,cool to 50° C. over 4 min, and hold at 50° C. for 4 min. The results(Table 25) showed that the peak and final viscosities were significantlylower in starch from the reduced SBEIIa grain compared to the controlwheat starch.

Starch Gelatinisation Properties.

Gelatinisation properties of starch were studied using differentialscanning calorimetry (DSC) as described in Regina et al., (2004). DSCwas carried out on a Perkin Elmer Pyris 1 differential scanningcalorimeter. Starch and water were premixed at a ratio of 1:2 andapproximately 50 mg weighed into a DSC pan which was sealed and left toequilibrate overnight. A heating rate of 10° C. per minute was used toheat the test and reference samples from 30 to 130° C. Data was analysedusing the software available with the instrument. The results (Table 26)clearly showed a delayed end of gelatinisation temperature (72.6° C.)for starch from the reduced SBEIIa grain compared to the control (66.6°C.). The peak gelatinisation temperature was also higher in the reducedSBEIIa starch (63.51° C.) compared to the control starch (61.16° C.).

Example 13: Analysis of High Amylose Wheat Flour During Processing

Pressure processing studies in collaboration with CSIRO Food andNutritional Sciences, Werribee.

Structural characterisation of high amylose wheat starches in comparisonwith native starch was carried out using Small Angle X-ray Scattering(SAXS). The study was designed to include a) characterising raw wheatflour and b) real-time analysis of the gelatinisation process whilepressure cooking the flour or starch samples at temperatures of greaterthan 100° C. and c) Structural changes on cooling over a period of 0 to10 days, and retrogradation. The study used wheat flour samples ofvarying amylose content ranging from about 25% (wild-type) to about 75%,increasing in increments of about 10%.

Three sets of flour samples were included in the experiments. Firstly,with pure lines without pooling from a high amylose wheat from thereduced SBEIIa lines, a medium level amylose wheat line AC45.1 which wastransformed with the hp-combo construct having about 50% amylose(Example 2) and from the control wheat (NB1). Secondly, with pooledwheat material from transformed lines as described in Example 2, poolingsamples in increments of 10% increasing amylose content. Thirdly,comparing flour from different species including wheat (high amylose,wild-type, and wheat lacking SSIIa), barley (wild-type, high amylose byreduced SBEIIa and SBEIIb, and high amylose by reduced SSII), and highamylose maize. The results from the resistant starch analysis on thepooled wheat material with a range of amylose content revealed a linearincrease in resistant starch from an amylose content of ≥40%.

Example 14: Production of Breads and Other Food Products

One of the most effective ways of delivering a grain such as highamylose wheat into the diet is through bread. To show that the highamylose wheat could readily be incorporated into breads and to examinethe factors that allowed retention of bread making quality, samples offlour were produced, analysed and used in baking. The following methodswere employed.

Methods.

Wheat grains were conditioned to 16.5% moisture content overnight andmilled with either a Buhler laboratory scale mill at BRI Ltd, Australia,or using a Quadromat Junior mill followed by sieving, to achieve a finalparticle size of 150 μm. The protein and moisture content of the sampleswere determined by infrared reflectance (NIR) according to AACC Method39-11 (1999), or by the Dumas method and air-oven according to AACCMethod 44-15 A (AACC₅ 1999).

Micro Z-Arm Mixing.

Optimum water absorption values of wheat flours were determined with theMicro Z-arm Mixer, using 4 g of test flour per mix (Gras et al., (2001);Bekes et al., (2002). Constant angular velocity with shaft speeds forthe fast and slow blades of 96 and 64 rpm, respectively, were usedduring all mixes. Mixing was carried out in triplicate, each for 20minutes. Before adding water to the flour, the baseline wasautomatically recorded (30 sec) by mixing only the solid components. Thewater addition was carried out in one step using an automatic waterpump. The following parameters were determined from the individualmixing experiments by taking the averages: WA %—Water Absorption wasdetermined at 500 Brabender Unit (BU) dough consistency; DoughDevelopment Time (DDT): time to peak resistance (sec).

Mixograms.

To determine optimal dough mixing parameters with the modified wheatflour, samples with variable water absorption corresponding to waterabsorption determined by the Micro Z-arm mixer, were mixed in a 10 gCSIRO prototype Mixograph keeping the total dough mass constant. Foreach of the flour samples, the following parameters were recorded:MT—mixing time (sec); PR—Mixograph peak resistance (Arbitrary Units,AU); BWPR—band width at peak resistance (Arbitrary Units, AU);RBD—resistance breakdown (%); BWBD—bandwidth breakdown (%); TMBW—time tomaximum bandwidth (s); and MBW—maximum bandwidth (Arbitrary Units,A.U.).

Micro Extension Testing.

Dough extensibility parameters were measured as follows: Doughs weremixed to peak dough development in a 10 g prototype Mixograph. Extensiontests at 1 cm/s were carried out on a TA.XT2i texture analyser with amodified geometry Kieffer dough and gluten extensibility rig (Mann etal., 2003). Dough samples for extension testing (˜1.0 g/test) weremoulded with a Kieffer moulder and rested at 30° C. and 90% RH for 45min. before extension testing. The R_Max and Ext_Rmax were determinedfrom the data with the help of Exceed Expert software (Smewing, Themeasurement of dough and gluten extensibility using the SMS/Kieffer rigand the TA.TX2 texture analyzer handbook, SMS Ltd: Surrey, UK, 1995;Mann, (2002).

An illustrative recipe based on the 14 g flour as 100% was as follows:flour 100%, salt 2%, dry yeast 1.5%, vegetable oil 2%, and improver(ascorbic acid 100 ppm, fungal amylose 15 ppm, xylanase 40 ppm, soyflour 0.3%, obtained from Goodman Fielder Pty Ltd, Australia) 1.5%. Thewater addition level was based on the micro Z-arm water absorptionvalues that were adjusted for the full formula. Flour (14 g) and theother ingredients were mixed to peak dough development time in a 35 gMixograph. The moulding and panning was carried out in a two stagedproofing steps at 40 C at 85% RH. Baking was carried out in a Rotel ovenfor 15 min at 190° C. Loaf volume (determined by the canola seeddisplacement method) and weight measurements were taken after cooling ona rack for 2 hours. Net water loss was measured by weighing the loavesover time.

The flour or wholemeal may be blended with flour or wholemeal fromnon-modified wheats or other cereals such as barley to provide desireddough and bread-making or nutritional qualities. For example, flour fromcvs Chara or Glenlea has a high dough strength while that from cv Janzhas a medium dough strength. In particular, the levels of high and lowmolecular weight glutenin subunits in the flour is positively correlatedwith dough strength, and further influenced by the nature of the allelespresent.

Flour from transgenic wheat lines having reduced SBEIIa were used at100%, 60% and 30% addition levels. e.g. either all the flour came fromthe various wheat lines or 60% or 30% were added to the Baking Control(B. extra) flour. Percentages are of total flour in the breadformulation. Four transgenic wheat lines were used as follows: 072(reduced SBEIIa), 212 (a wheat line derived from the cross, reducedSBEIIa×SBEI triple null wheat), H7 (a wheat line derived from the cross,reduced SBE IIa×SSIIa triple null wheat) and 008 (reduced SBEIIb) weretested along with a non transformed control wheat (NB1). All wheats weremilled in a Brabender Quadramat Junior mill. All blends had waterabsorptions determined on 4 g Z-arm mixer and optimal mixing timesdetermined on 10 g Mixograph as described above. These conditions wereused in preparing the 10 g test bake loaves.

Mixing Properties.

Apart from the control lines (Baking Control, NB1 and 008) all otherwheat lines gave greatly elevated water absorption values (FIG. 17A).Lines 212 and 072 gave increasing water absorption values withincreasing addition levels, including up to a high of 95% waterabsorption at 100% addition of 212 flour. Increased incorporation levelsof flour from these lines also lead to a decrease in the optimalMixograph mixing times (FIG. 17B). As with the water absorption data,the non-control lines showed a strong reduction in specific loaf volume(loaf volume/loaf weight) with increasing levels of addition. The effectwas particularly strong for the 212 line.

These studies show that breads with commercial potential, includingacceptable crumb structure, texture and appearance, could be obtainedusing the high amylose wheat flour blended with control flour samples.Furthermore, high amylose wheats are used in combination with preferredgenetic background characteristics (e.g. preferred high and lowmolecular weight glutenins), the addition of improvers such as gluten,ascorbate or emulsifiers, or the use of differing bread-making styles(e.g. sponge and dough bread-making, sour dough, mixed grain, orwholemeal) to provide a range of products with particular utility andnutritional efficacy for improved bowel and metabolic health.

Other Food Products:

Yellow alkaline noodles (YAN) (100% flour, 32% water, 1% Na₂CO₃) wereprepared in a Hobart mixer using the standard BRI Research NoodleManufacturing Method (AFL 029). Noodle sheet was formed in the stainlesssteel rollers of an Otake noodle machine. After resting (30 min) thenoodle sheet was reduced and cut into strands. The dimensions of thenoodles were 1.5×1.5 mm.

Instant noodles (100% flour, 32% water, 1% NaCl and 0.2% Na2CO3) wereprepared in a Hobart mixer using the standard BRI Research NoodleManufacturing method (AFL 028). Noodle sheet was formed in the stainlesssteel rollers of an Otake noodle machine. After resting (5 min) thenoodle sheet was reduced and cut into strands. The dimensions of thenoodles were 1.0×1.5×25 mm. The noodle strands were steamed for 3.5 minand then fried in oil at 150 C for 45 sec.

Sponge and Dough (S&D) bread. The BRI Research sponge and dough bakinginvolved a two-step process. In the first step, the sponge was made bymixing part of the total flour with water, yeast and yeast food. Thesponge was allowed to ferment for 4 h. In the second step, the spongewas incorporated with the rest of the flour, water and other ingredientsto make dough. The sponge stage of the process was made with 200 g offlour and was given 4 h fermentation. The dough was prepared by mixingthe remaining 100 g of flour and other ingredients with the fermentedsponge.

Pasta—Spaghetti. The method used for pasta production was as describedin Sissons et al., (2007). Test sample flours from high amylose wheat(reduced SBEIIa) and control wheat (NB1) were mixed with Manildrasemolina at various percentages (test sample: 0, 20, 40, 60, 80, 100%)to obtain flour mixes for small scale pasta preparation. The sampleswere corrected to 30% moisture. The desired amount of water was added tothe samples and mixed briefly before being transferred into a 50 gfarinograph bowl for a further 2 min mix. The resulting dough, whichresembled coffee-bean-size crumbs, was transferred into a stainlesssteel chamber and rested under a pressure of 7000 kPa for 9 min at 50 C.The pasta was then extruded at a constant rate and cut into lengths ofapproximately 48 cm. Two batches of pasta were made for each sample. Thepasta was dried using a Thermoline Temperature and Humidity Cabinet (TEC2604) (Thermoline Scientific Equipment, Smithfield, Australia). Thedrying cycle consisted of a holding temperature of 25 C followed by anincrease to 65 C for 45 min then a period of about 13 h at 50 C followedby cooling to 25 C. Humidity was controlled during the cycle. Driedpasta was cut into 7 cm strands for subsequent tests.

Example 15: In Vitro Measurements of Glycaemic Index (GI) and ResistantStarch (RS) of Food Samples

The Glycemic Index (GI) of food samples including the bread made asdescribed herein was measured in vitro as follows: Food samples werehomogenised with a domestic food processor. An amount of samplerepresenting approximately 50 mg of carbohydrate was weighed into a 120ml plastic sample container and 100 μl of carbonate buffer added withoutα-amylase. Approximately 15-20 seconds after the addition of carbonatebuffer, 5 ml of Pepsin solution (65 mg of pepsin (Sigma) dissolved in 65ml of HCl 0.02M, pH 2.0, made up on the day of use) was added, and themixture incubated at 37° C. for 30 minutes in a reciprocating water bathat 70 rpm. Following incubation, the sample was neutralised with 5 ml ofNaOH (0.02M) and 25 ml of acetate buffer 0.2M, pH 6 added. 5 ml ofenzyme mixture containing 2 mg/mL of pancreatin (α-amylase, Sigma) and28 U/mL of amyloglucosidase from Aspergillus niger (AMG, Sigma)dissolved in Na acetate buffer (sodium acetate buffer, 0.2 M, pH 6.0,containing 0.20 M calcium chloride and 0.49 mM magnesium chloride) wasthen added, and the mixture incubated for 2-5 minutes. 1 ml of solutionwas transferred from each flask into a 1.5 ml tube and centrifuged at3000 rpm for 10 minutes. The supernatant was transferred to a new tubeand stored in a freezer. The remainder of each sample was covered withaluminium foil and the containers incubated at 37° C. for 5 hours in awater bath. A further 1 ml of solution was then collected from eachflask, centrifuged and the supernatant transferred as carried outpreviously. This was also stored in a freezer until the absorbancescould be read.

All samples were thawed to room temperature and centrifuged at 3000 rpmfor 10 minutes. Samples were diluted as necessary (1 in 10 dilutionusually sufficient), 10 μl of supernatant transferred from each sampleto 96-well microtitre plates in duplicate or triplicate. A standardcurve for each microtitre plate was prepared using glucose (0 mg, 0.0625mg, 0.125 mg, 0.25 mg, 0.5 mg and 1.0 mg). 200 μl of Glucose Trinderreagent (Microgenetics Diagnostics Pty Ltd, Lidcombe, NSW) was added toeach well and the plates incubated at room temperature for approximately20 minutes. The absorbance of each sample was measured at 505 nm using aplate reader and the amount of glucose calculated with reference to thestandard curve.

The level of Resistant Starch (RS) in food samples including the breadmade as described herein was measured in vitro as follows. This methoddescribes the sample preparation and in vitro digestion of starch infoods, as normally eaten. The method has two sections: firstly, starchin the food was hydrolysed under simulated physiological conditions;secondly, by-products were removed through washing and the residualstarch determined after homogenization and drying of the sample. Starchquantitated at the end of the digestion treatment represented theresistant starch content of the food.

On day 1, the food samples were processed in a manner simulatingconsumption, for example by homogenising with a domestic food processorto a consistency as would be achieved by chewing. After homogenising, anamount of food representing up to 500 mg of carbohydrate was weighedinto a 125 mL Erlenmeyer flask. A carbonate buffer was prepared bydissolving 121 mg of NaHCO₃ and 157 mg of KCl in approximately 90 mLpurified water, adding 159 μL of 1 M CaCl₂.6H₂O solution and 41 μL of0.49 M MgCl₂.6H₂O, adjusting the pH to 7 to 7.1 with 0.32 M HCl, andadjusting the volume to 100 mL. This buffer was stored at 4° C. for upto five days. An artificial saliva solution containing 250 units ofα-amylase (Sigma A-3176 Type VI-B from porcine pancreas) per mL of thecarbonate buffer was prepared. An amount of the artificial salivasolution, approximately equal to the weight of food, was added to theflask. About 15-20 sec after adding the saliva, 5 mL of pepsin solutionin HCl (1 mg/mL pepsin (Sigma) in 0.02 M HCl, pH 2.0, made up on day ofuse) was added to each flask. The mixing of the amylase and then pepsinmimicked a human chewing the food before swallowing it. The mixture wasincubated at 37° C. for 30 min with shaking at 85 rpm. The mixture wasthen neutralised with 5 mL of 0.02M NaOH. 25 mL of acetate buffer (0.2M, pH 6) and 5 mL of pancreatin enzyme mixture containing 2 mg/mLpancreatin (Sigma, porcine pancreas at 4×USP activity) and 28 U ofamyloglucosidase (AMG, Sigma) from Aspergillus niger in acetate buffer,pH6, were added per flask. Each flask was capped with aluminium foil andincubated at 37° C. for 16 hours in a reciprocating water bath set to 85rpm.

On day 2, the contents of each flask were transferred quantitatively toa 50 mL polypropylene tube and centrifuged at 2000×g for 10 min at roomtemperature. The supernatants were discarded and each pellet washedthree times with 20 mL of water, gently vortexing the tube with eachwash to break up the pellet, followed by centrifugation. 50 μL of thelast water wash was tested with Glucose Trinder reagent for the absenceof free glucose. Each pellet was then resuspended in approximately 6 mLof purified water and homogenised three times for 10 seconds using anUltra Turrax TP18/10 with an S25N-8G dispersing tool. The contents arequantitatively transferred to a 25 mL volumetric flask and made tovolume. The contents were mixed thoroughly and returned to thepolypropylene tube. A 5 mL sample of each suspension was transferred toa 25 mL culture tube and immediately shell frozen in liquid nitrogen andfreeze dried.

On day 3, total starch in each sample was measured using reagentssupplied in the Megazyme Total Starch Procedure kit. Starch standards(Regular Maize Starch, Sigma 5-5296) and an assay reagent blank wereprepared. Samples, controls and reagent blanks were wet with 0.4 mL of80% ethanol to aid dispersion, followed by vortexing. Immediately, 2 mLof DMSO was added and solutions mixed by vortexing. The tubes wereplaced in a boiling water bath for 5 min, and 3 mL of thermostableα-amylase (100 U/ml) in MOPS buffer (pH 7, containing 5 mM CaCl₂) and0.02% sodium azide) added immediately. Solutions were incubated in theboiling water bath for a further 12 min, with vortex mixing at 3 minintervals. Tubes were then placed in a 50° C. water bath and 4 mL ofsodium acetate buffer (200 mM, pH 4.5, containing 0.02% sodium azide)and 0.1 mL of amyloglucosidase at 300 U/ml added. The mixtures wereincubated at 50° C. for 30 min with gentle mixing at 10 min intervals.The volumes were made up to 25 mL in a volumetric flask and mixed well.Aliquots were centrifuged at 2000×g for 10 min. The amount of glucose in50 μL of supernatant was determined with 1.0 mL of Glucose Trinderreagent and measuring the absorbance at 505 nm after incubation of thetubes at room temperature in the dark for a minimum of 18 min and amaximum of 45 min.

Bread loaves baked from flour from four transgenic wheat lines, namely072 (reduced SBEIIa), 212 (a wheat line derived from the cross, reducedSBEIIa×SBEI triple null wheat), H7 (a wheat line derived from the cross,reduced SBEIIa×SSIIa triple null wheat) and 008 (reduced SBEIIb) weretested along with a non transformed control wheat (NB1) for RS and GIafter incorporation levels of 100%, 60% and 30% flour, the remainder 40%or 70% flour being from wild-type grain. Increased incorporation of 212,072, and H7 flour resulted in significant increases in RS (FIG. 18A andreductions in predicted GI (FIG. 18B). The magnitude of the changes wasgreatest when using flour from Line 212. For instance, bread made with100% addition of this high amylose flour had an RS content of about 10%which represented a 150% increase above that for 30% level of inclusionand a 9-fold increase compared to the NB1 controls. Increasing theextent of incorporation of flour from the 008 lines had no effect on theRS and GI of the resultant loaves and the results were comparable tothose of the baking control flour.

Example 16: Processing of High Amylose Wheat and Resultant RS Levels

A small scale study was conducted to determine the resistant starch (RS)content in processed grain from the high amylose wheat which had beenrolled or flaked. The technique involved conditioning the grains to amoisture level of 25% for one hour, followed by steaming the grains.Following steaming, the grains were flaked using a small-scale roller.The flakes were then roasted in an oven at 120 C for 35 min. Two rollerwidths and three steaming timings were used on approximately 200 g ofsamples from high amylose wheat having reduced SBEIIa (HAW, line 85.2c)and wild-type, control wheat (cv. Hartog). The roller widths tested were0.05 mm and 0.15 mm. The steaming timings tested were 60′, 45′ and 35′.

This study showed a clear and substantial increase in the amount of RSin processed high amylose wheat compared to the control (Table 27, FIGS.18A and 18B). There also appeared to be some effect of the processingconditions on the RS level. For example with the high amylose grain,increased steaming times led to a slight reduction in the level of RS,most likely due to increased starch gelatinization during steaming(Table 27). The wider roller gap generated a higher RS level except atthe longest steaming time. This could have been due to increased sheardamage of the starch granules when the grains were rolled at narrowergaps, reducing RS levels slightly. Narrower roller gaps also led tohigher RS levels in the Hartog control, albeit at much lower overall RSlevels. In contrast to the high amylose results, increased steamingtimes led to higher RS levels, possibly due to increased starchgelatinization at longer steaming times contributing to more starchretrogradation during subsequent processing and cooling.

Consolidated data on RS from various products. RS data obtained fromvarious products such as noodles, sponge and dough bread and spaghetti,prepared as described in Example 10, are presented in Table 28. Not alllevels of incorporation were tested for all products, but incorporationlevels of 20%, 40% and 60% were used in most of the products analysed.The results showed a linear relationship between RS content and thelevel of incorporation of high amylose flour.

Example 17: Isolation of Plants Having Point Mutations in SBEIIa

A population of mutated plant lines was developed after EMS mutagenesisof seeds of the wheat cultivars Arche or Apache, using standard EMStreatment conditions. About 5000 Apache and 900 Arche individual M1plants were grown from the mutagenised seed, self-fertilised, and seedsfrom each plant and subsequent progeny generations maintained aspotentially mutant lines, each derived from an individual M1 plant. Thelines were screened for mutations in the three homoeologous SBEIIa genesby next-generation Solexa sequencing (Illumina). To do this, 7 DNA poolswere prepared, each by pooling DNA from about 130 M1 families from theArche population and 96 from the Apache population. PCR was carried outon the pooled DNAs for 3 or 4 regions per homoeologous gene, targetingthe exonic regions including splice sites of the genes. Genome-specificprimers are set out in Table 29.

The 10 amplicons (amplification products) from the same DNA pools weremerged after normalization of the PCR products, and sequencing was donewith one flow cell per DNA pool. The sequence data were analysed toselect from all of the polymorphisms the ones most likely due tomutations rather than to sequencing errors, based on the frequencies ofthe observed polymorphisms. 64 putative mutants from the Archepopulation and 48 from the Apache population were observed from thefirst sequence analysis covering the exonic regions and splice sites.SNP assays were designed for each polymorphism based on kaspartechnology, and genotyping was performed on the 130 families in eachpool that was positive for the particular polymorphism. Thereby, theindividual mutant line containing each mutant gene was identified andthe mutant SBEIIa sequences confirmed.

By this method, 31 mutant lines from the Apache population and 9 fromthe Arche population were identified each having an SBEIIa mutation, andM2 kernels of each retained. From each mutant line, depending onavailability, around 10 M2 seeds, were cut in half, the half without theembryo was used for DNA extraction and analysis, the other half with theembryo was saved for sowing. A total of 5 mutants were confirmed on halfseeds from Arche population and 28 from Apache population. Thecorresponding seeds were sown to produce progeny plants to confirm thatthe mutations were inherited in Mendelian fashion by repeating analysison M2 plant leaf material, providing much better DNA quality. Theseanalyses confirmed 19 mutants, 4 from the Arche population and 15 fromthe Apache population and allowed their ranking depending on their DNAand the deduced protein sequences encoded by the mutants.

The obtained mutants included ones which had mutated SBEIIa genes withstop codons in the protein coding regions of the SBEIIa genes on the Bor D genomes, causing premature termination of translation of the SBEIIaproteins, and lines with splice site mutations in the SBEIIa-B or -Dgenes. Such mutations were expected to be null mutations. Pointmutations in the SBEIIa A, SBEIIa-B and SBEIIa-D genes such as aminoacid substitution mutations were also obtained and their impact on thestructure of the encoded proteins predicted using Blosum 62 and Pam 250matrices.

Plants from the most promising 8 mutant lines were crossed withdouble-null SBEIIa mutants of the appropriate genotype including A1D2,A2D2, A1B2, B2D2 genotypes in order to produce the triple-mutant plantsand seed in the F2 generation. Fertile plants producing seeds with atleast 50% amylose in the starch content are selected. Mutant plants werealso crossed with durum wheat (cultivar Soldur) to introduce themutations into the tetraploid wheat.

TABLE 1 Starch branching enzyme genes characterized from cereals SBEType of Species isoform clone Accession No. Reference Maize SBEI cDNAU17897 Fisher et al., Journal Plant Physiol. 108(3): 1313-1314, 1995genomic AF072724 Kim et al., Gene. 216 (2): 233-43, 1998a SBEIIb cDNAL08065 Fisher et al., Plant Physiol 102: 1045-1046, 1993 genomicAF072725 Kim et al., Plant Physiol. 121(1): 225-236, 1999 SBEIIa cDNAU65948 Gao et al., 1997 Wheat SBEII cDNA Y11282 Nair et al., Plant Sci122: 153-163, 1997 SBEI cDNA and AJ237897 (SBEI gene) Baga et al., PlantMol genomic AF002821 (SBEI Biol. 40(6): 1019-1030, pseudogene 1999AF076680 (SBEI gene) Rahman et al., Genome AF076679 (SBEI cDNA) 40:465-474, 1997, Rahman et al., 1999 SBEI cDNA Y12320 Repellin et al.,Plant Gene Reg pp. 97-094, 1997 SBEIIa cDNA and AF338432 (cDNA) Rahmanet al., 2001 genomic AF338431 (gene) SBEIIa cDNA AK335707, AF286319SBEIIb cDNA and WO 01/62934 genomic SBEIIb cDNA WO 00/15810 SBEIIb-DcDNA US2005074891 Rice SBEI cDNA D10752 Nakamura, 2002 and Nakamura andYamanouchi, Plant Physiol. 99(3): 1265-1266, 1992 SBEI genomic D10838Kawasaki et al., Mol Gen Genet. 237(1-2): 10-6, 1993 RBE3 cDNA D16201Mizuno et al., 1993 Barley SBEIIa and cDNA and AF064563 (SBEIIb gene)Sun et al., 1998 SBEIIb genomic AF064561 (SBEIIb cDNA) AF064562 (SBEIIagene) AF064560 (SBEIIa cDNA)

TABLE 2 Amino acid sub-classification Sub-classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Residues Tryptophan, Tyrosine, Phenylalanine that influenceGlycine and Proline chain orientation

TABLE 3 Exemplary and Preferred Conserved Amino Acid SubstitutionsExemplary conservative Preferred conservative Original Residuesubstitutions substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn, His, Lys,Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu,Val, Met, Ala, Phe Leu Leu Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, AsnArg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser ThrThr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu,Met, Phe, Ala Leu

TABLE 4 Genome specific primers for wheat SBEIIa genes Expected GenomePrimers Region Size (bp) A SbeIIa_A_deb1F/SbeIIa_A_deb1R Exons 1 to 8615 A SbeIIa_A_deb2F/SbeIIa_A_deb1R Exons 1 to 8 604 ASbeIIa_A_deb2F/SbeIIa_A_deb5R Exons 1 to 8 ~1039 ASbeIIa_A_deb3F/SbeIIa_A_deb1R Exons 1 to 8 565 ASbeIIa_A_deb4F/AR2aE8R07 Exons 1 to 8 735 A SbeIIa_A_deb5F/AR2aE8R07Exons 1 to 8 696 B SbeIIa_B_R4/BeIIaE1f Exons 1 to 8 ~600 on B, ~800 onA D SbeIIa_D_deb1F/SbeIIa_D_deb1R Exons 1 to 8 573 DSbeIIa_D_deb1F/SbeIIa_D_deb2R Exons 1 to 8 539 DSbeIIa_D_deb1F/SbeIIa_D_deb4R Exons 1 to 8 ~900 DSbeIIa_D_deb2F/SbeIIa_D_deb4R Exons 1 to 8 ~900 DSbeIIa_D_deb3F/SbeIIa_D_deb4R Exons 1 to 8 ~900 DSbeIIa_D_deb4F/AR2aE8R07 Exons 1 to 8 736 A Snp1for/Arev5 Exons 13-14508 A Afor4/del4rev Exons 12-14 863 A Snp6for/Arev5 Exon 14 205 AAfor4/Snp6rev Exons 12-13 637 A Afor4/del5rev Exons 12-14 872 BBsnp4/Arev5 Exons 13-14 494 B Afor4/Bsnp17rev Exons 12-14 905 BAfor4/Bsnp18rev Exons 12-14 952 D Afor4/Dsnp7rev Exons 12-14 901 DDsnp7for/Drev1 — 278 D Afor4/Arev5 Exons 12-14 802

TABLE 5 Nucleotide sequences of genome specific primers of SBEIIa SEQNucleotide Sequence ID Primer name (5′ to 3′) NO: SbeIIa_A_deb1FGTTCGATGCTGTTCCCCAG 36 SbeIIa_A_deb1R AGCCGTTTGCTCCTCGATG 37SbeIIa_A_deb2F TTCCCCAGTTGATCTCCATC 38 SbeIIa_A_deb4FCTTACTGAATACTGACCAGTTG 39 SbeIIa_A_deb5F TTTATGATCTGGCTTTTGCATCCTA 40SbeIIa_A_deb5R GATGTTCCCCAAATTTGCATGAC 41 SbeIIa_B_deb4RAATGCACAAGGCAGTGAAGTAG 42 SbeIIa_D_deb1F CCCAATTGATCTCCATGAGT 43SbeIIa_D_deb1R AACCCCAAACGGTGCATTATG 44 SbeIIa_D_deb2FCGGCTTTGATCATTCCTCG 45 SbeIIa_D_deb2R GCTAGAATGCACATCCATCTGAT 46SbeIIa_D_deb3F GTAACTGCAAGTTGTGGCG 47 SbeIIa_D_deb4FGCTTACTGAATACTGACCAGTTACTA 48 SbeIIa_D_deb4R CCTTAATTCAAAATGAGCGAAAGC 49snp1for GGCTAACTGTTCCTGTTAAA 50 snp6for GATGAGATCATGGACGATTC 51 snp6revAATAAATAATAATCACTTCG 52 Del4rev GAGTAACAGCCTGATCCCAA 53 Del5revTAACAAAAAGAGTAACAGCC 54 Bsnp4 GTCAATCTGTTCTTACACG 55 Bsnp17 revCAAAAAGAGTAGTAACAGCT 56 Bsnp18 rev CAAGGTATAAATTAGCATTC 57 D snp7 forGTTTTATTTTGGGGATCAGT 58 D snp7 rev CCCTAACAAAAAGTGTAACAGA 59 Afor4ATCAGACCTTGTCACCAAAT 60 Arev5 GCACTTACATCTTCACCAATG 61 Drev 1GCCTTCTGAAGCAATTGACAAG 62

TABLE 6 Primers designed to amplify parts of theSBEIIa gene specifically from the A genomeof wheat-detected polymorphisms and fragment sizes SEQ Primer ID codePrimer sequence SNP details Afor4 Arev5 Arev6 NO: snp1forGGCTAACTGTTCCTGTTAAA extra A/B and D 508 63 SNP1REV CGACATGTGTAAGAACAGATextra A/B and D 334 64 snp2for2a GTCGATATTCTATTCTTATGTt/D; a/B; a/B; c/B D 474 65 snp3for CTTTTTTAGGGCACTGAAAT c/B; c/B; c/B D315 66 snp3reva GTTATGATGCATAGCAATTA c/B D 528 67 snp4forTCTTAGATAGTTCCCTAGTAC t/B D 245 68 snp4rev CAGGTAAAATTGTACAAGCG t/B D599 69 snp5for ACCTGATGAGATCATGGAC a/B D 210 70 snp5for2TACCTGATGAGATCATGGAC a/B D 211 71 snp6for GATGAGATCATGGACGATTCa/B D; g/B D 205 72 snp6rev AATAAATAATAATCACTTCG t/B; a/B; g/B; g/B D637 73 snp7for TCTTTTTGTTAGGGGTAAG 3 first bp extra/D; 390 74extra act in BD; a/B D A for3 AGTTTGACCAAGTCTACTG 1050 75 Afor4ATCAGACCTTGTCACCAAAT t/D 802 76 Arev5 GCACTTACATCTTCACCAATG 802 77 Arev7GTAGTTATAAGCAATATG 78 del1for CATCAAGTGGTTTCAGTAAC 7 bp Difference/BD334 79 del1rev GTTACTGAAACCACTTGATG 490 80 Del4for TTGGGATCAGGCTGTTACTCextra g in B D; t = a  BD; 81 extra act in BD Del4revGAGTAACAGCCTGATCCCAA 863 82 Del5for GGCTGTTACTCTTTTTGTTAt/BD; extra t; act extra in BD; 83 extra ct Del5rev TAACAAAAAGAGTAACAGCC872 84 Del3for TTAACCAGTTAAGTAGTT extra cagt; extra a; extra ttaag 43285 in D and ttaatag in B Del3rev1 AACTACTTAACTGGTTAAextra ttaag in D and ttaatag in B; 836 86 extra a; extra actg Del3rev2GATCCCAAAATAAAACTACTT extra ttaag in D and ttaatag in B; 851 87 extra aDel3rev3 CCCAAAATAAAACTACTT extra ttaag in D and ttaatag in B; 848 88extra a

TABLE 7 Primers designed to amplify parts of theSBEIIa gene specifically from the B genome of wheat-detected polymorphisms and fragment sizes SEQ Primer ID codePrimer sequence SNP details Arev5 Afor4 Exons NO: Bsnp1forGTGGGATTCTCGTCTG a/A D 89 Bsnp2 TTGGGAAGTATGTAGCTGC ct/A D 546 13_14 90Bsnp3 TTGGCTAACTGTTCCTGTC t/A D 509 13_14 91 Bsnp4 GTCAATCTGTTCTTACACGt/A D; extra a in A; a/A D 494 92 Bsnp5 ATCTGTTCTTACACGTGTCAa/A D; t/D; g/A D 494 93 Bsnp6 GTCAATATTCTATTCTTATA t/D; g/A D; g/A D474 94 Bsnp7 CTATTCTTATACAGGTATTA g/A D; g/A D 465 95 Bsnp8AACGCGAGATGGTGGCTTGAT a/A D 430 half 96 13_14 Bsnp9 CAAGTGGTTTCAGTAACTTCt/A D 331 14 97 Bsnp10 TGGTTTCAGTAACTTCTTC t/A D; t/A D 327 98 Bsnp11GGAAGATTGGAAGTGATTG c/A; c/A; a/A D 195 14 99 Bsnp13 TGGAAGTGATTGTTATTATa/A D; ta/A D 188 100 Bsnp14 TTGCTTCTTGTTCTAGATGG t/D; a/A D 155 101Bsnp1rev TTCCCAACTCCCATAGTGAAC a/A D 290 half 102 12 Bsnp2revCAAATATGGTGACAGAAGTCG tc/A D 322 103 Bsnp3rev CACGTGTAAGAACAGATTGa/A D; extra a in A; t/A D 356 104 Bsnp4rev AGAATAGAATATTGACACg/A D; t/D; g/A D 371 105 Bsnp6rev GTAAGAATCTTAATACCTGT g/A D; g/A D 396106 B snprev7 CGCGTTTGACAGTAAGAATCTT g/A D 405 13 107 Bsnp8revCCATCAAACTTATATTCA a/A D 437 108 Bsnp9rev CAATTGTTTCAGTGCCCTGAAGt/A; t/A D; t/A D 539 12_13 109 Bsnp10rev GCAATTGTTTCAGTGCCCTGt/A; t/A D 540 110 Bsnp11rev CTTAGAAGAAAAAATAATAAC c/D; ta/A D; a/A D673 12_13 111 Bsnp13rev GCAAACTTAGAAGAAAAAA t/D; c/D; a/A D 678 112Bsnp14rev CCATAGTTCCCAGTAAATGC a/A D 713 12_13 113 Bextra1revCTACTATTAAATTAACTG ct extra/A, at extra/AD, 868 12_14 114taa extra/A, g/AD, actg extra/D Bsnp16 rev ATCCCCAAAATAAAACTACTATc extra/A, tat extra /AB 880 12_14 115 Bsnp17 rev CAAAAAGAGTAGTAACAGCTag extra/D, agt extra/A, 905 12_14 116 a extra/D, t/D, g/AD Bsnp18 revCAAGGTATAAATTAGCATTC c/AD 952 12_14 117 Bsnp19 rev GCATTCTTATGAAAAGACc/AD, c/AD 938 12_14 118

TABLE 8 Primers designed to amplift parts of theSBEIIa gene specifically from the D genome ofwheat--detected polymorphisms and fragment sizes SEQ ID Primer codePrimer sequence SNP details Arev5 Drev 1 Afor4 NO: D snp1forTCTGTTCTTACACATGTT c/A B 489 798 119 D snp1for/A CTTTTTTAGGGCACTGAAACc/B; c/B; t/A 315 624 120 Dsnp2 for GATTATTATTTATTTTCCTTCTAAg/B; at/B; t/AB; cAB 184 490 121 GTTTGT Dsnp2bforACCTGATGAGATCATGGAAGATTG c/A; c/A 210 519 122 D snp 3 forGTGATTATTATTTATTTTC g/B; at/B; t/AB; cAB 183 492 123 D snp 4 forTTATTTTCCTTCTAAGTTTGT at/B; t/AB; c/AB 172 481 124 D snp5forGTGATTATTATTTATTTTC g/B; at/B; t/AB 137 446 125 D snp6forTGATGCGGTAGTTTACTTGATGT g/B; a/B; c/AB 89 398 126 D del1forGATTTTTAACTAGTTAAGTAGTT t/B; cagt/AB; a/AB; 298 127 t/B; at/B; del in AD snp7 for GTTTTATTTTGGGGATCAGT del g in A; a/B; g/AB 278 128 D snp1 revCCTGCATAAGAATAGAATATCA t/A; a/B; c/AB 379 129 D snp1a revCATGTTATGATGCATAGCAATTG t/A 556 130 D snp2 rev GTAAATGTCATCTAGAACAAGAAAg/B; c/AB 701 131 D snp3 rev CAAGAAACAAACTTAGAAGG c/AB; t/AB 684 132D snp4 rev ACAAACTTAGAAGGAAAATAA c/AB; t/AB; at/B 678 133 D snp5 revCATCAGTAGCAAATCCAAAATAT g/AB 739 134

TABLE 9 Genome specific primers for wheat SBEIIb genes Genome PrimersExpected Size (bp) A SbeIIb_A_deb1F/2R 741 A SbeIIb_A_deb1F/4R 1007 ASbeIIb_A_deb4F/4R 772 B SbeIIb_B_deb3F/2R 615 B SbeIIb_B_deb2F/3R 929 BSbeIIb_B_deb3F/4R 772 D SbeIIb_D_deb1F/1R 1126 D SbeIIb_D_deb3F/3R 827 DSbeIIb_D_deb4F/4R 669

TABLE 10 Nucleotide sequences of genome specific primers of SBIIHb SEQNucleotide Sequence ID Primer name (5′ to 3′) NO: SbeIIb_A_deb1FACCCCGTAATTATTGGCGCT 135 SbeIIb_A_deb4F ACTCTGATGATCTGAAGGTAG 136SbeIIb_A_deb2R TCATGCAGGCAGGTACTAG 137 SbeIIb_A_deb4RGTGGCAGAATGCGTAATTTCTCT 138 SbeIIb_B_deb2F CAGCGATCTTACGTTCCCTA 139SbeIIb_B_deb3F ATGTCTGTAGGTGCCGTCA 140 SbeIIb_B_deb2RCAACAAATTAGAAAGAGGATATTCC 141 SbeIIb_B_deb3R CCGTAGATGATTCTTTGTCCATTA142 SbeIIb_B_deb4R ATGGAACCTAACACAATGTGC 143 SbeIIb_D_deb1FGCGCCACCTTTCTCACTCA 144 SbeIIb_D_deb3F CGGTCCCGTTCAGTTCGAT 145SbeIIb_D_deb4F CCTGAGTAAATACTGCCACCA 146 SbeIIb_D_deb1RAGAATGCGTAATTTCTCCCTCG 147 SbeIIb_D_deb3R TGTCTTCAGCATCAATTTCTTCAC 148SbeIIb_D_deb4R CTGTAGGCTTGTTTCATCATCA 149

TABLE 11 SBEII expression vs Amylose content of RNAi lines of wheatSBEIIa SBEIIb expression expression relative relative Total SBEIIselected Amylose to a WT to a WT expression line Construct % (%) (%) (%of WT) 673.2.1 hp-combo 35 108 91 100 679.5.3 hp-combo 40 81 1 41670.1.4 hp-combo 45 35 10 23 672.2.3 hp-combo 50 16 1 9 671.2.2 hp-combo55 8 5 7 666.2.2 hp-combo 60 10 6 8 669.1.2 hp-combo 65 9 7 8 684.2.3hpc-SBEIIa 70 6 10 8 677.1.2 hp-combo 75 4 1 3 684.2.1 hpc-SBEIIa 80 3 54 694.3.3 hpc-SBEIIa 85 2 3 3

TABLE 12 List of microsatellite markers tested in the mutants Chromosome2A Chromosome 2B Chromosome 2D gwm 304 barc 128 gwm 539 gwm 328 gwm 129cfd 270 barc 309 wmc 265 cfd 168 cfa 2043 wmc 272 cfd 233 cfa 2058 gwm388 wmc 175 wmc 170 wmc 441 wmc 181 gwm 312 barc 101 wmc 041 gwm 294 gwm120 cfd 239 wmc 181 gwm 130 gwm 349 gwm 356 gwm 526 barc 219 gwm 265 gwm501 gwm 382 wmc 181 wmc 332 wmc 167 gwm 311 wmc 434 gwm 320 gwm 382 wmc361 gwm 301 cfa 2086 gwm 382 cfd 50 wmc 317 barc 159 wmc 445

TABLE 13 Mutants identified from HIB population and microsatellitemapping data Microsatellite mapping (markers retained/markers Mutanttype Genome Mutant number tested) Type 1 A 20-257 (H7) 15/15 19-119 (G3) 5/11 12-178 10/10 5-563 10/10 21C-880D  4/10 B 12-679  7/15 5-173 15/1513-963 (F10)  4/11 18c-109 8/8 3-159 3/8 D 19-832 (A6) 13/13 22-578 (B5)13/13 3-909 (D1)  7/13 19b-918 (C11) To be done Type 2 A 20b-5B2-608(H2) 10/10 19c-342  9/10 19-744 12/12 B 21-142 (F6), 15/15 21-668 (D2-2)15/15 20-365 15/15 19-220 14/15 21b-4B2-345 (A8) 11/11 20-141  9/11 D12-801 13/13 5-706 13/13 19c-905 To be done 18b-505 To be done Type 3 A18-111/3 (D2-1)  8/11 19-861 (F9)  8/11 20-791 (G10) 12/12 B 19b-55 (G7)11/11 D 18-96 (E12) 18/18 18b-120 (E3) To be done 18b-190 (C12) To bedone

TABLE 14 Double null mutants of SBEII identified Number of double CrossParental lines Genotype of nulls designation (genotype of parent) doublenull identified 08/a 20-257 (A1) X 5-173 (B1) A1B1 6 08/b 20-257(A1) X19-832 (D1) A1D1 2 08/c 19-832 (D1) X 5-173 (B1) B1D1 0 08/d 21-142 (B2)X 12-801 (D2) B2D2 4 08/e 21-142 (B2) X 5-706 (D2) B2D2 8 08/f 20-365(B2) X 12-801(D2) B2D2 4 08/g 21-668 (B2) X 5-706 (D2) B2D2 6 08/h20-257 (A1) X 21-142 (B2) A1B2 2 08/i 20-257 (A1) X 12-801 (D2) A1D2 508/j 18-111/3 (A3) X 18-96 (D3) A3D3 2 08/k 18-111/3 (A3) X 5-173 (B1)A3B1 3 08/l 18-96 (D3) X 5-173 (B1) B1D3 1

TABLE 15 Crosses performed between double and single null mutants CrossParent 1 P1 Parent 2 P2 Potential designation Code genotype Codegenotype F2 genotype 08/aa 5-173 B1 08/b-18 A1D1 A1B1D1 08/aa-2 5-173 B108/b-33 A1D1 A1B1D1 08/bb 5-706 D2 08/h-92 A1B2 A1B2 D2 08/dd 5-706 D208/h-111 A1B2 A1B2 D2 08/ee 5-173 B1 08/b-12 A1D1 A1B1D1 08/ff 21-142 B2 08/b-12 A1D1 A1B2D1 08/gg 20-365  B2 08/b-12 A1D1 A1B2D1

TABLE 16 Amylose content in grain starch of progeny from crosses betweendouble null mutants and single null mutants Lines Genotype Amylose % HIBmutant F2 of triple null cross 67.38 Cadoux WT 35.4 85.2c hp-SBEIIa74.99 008 (IIb knock out) hp-SBEIIb 36.1 Chara WT 36.09

TABLE 17 Fertility observations on F2 progeny plants % fertile Number ofseed Line ID Genotype spikes per head 08/dd S28 A1D2(hetB2) 41.9 17.008/dd S14 A1B2(hetD2) 75.3 26.3 08/dd S22 A1D2(hetB2) 56.5 19.0 08/ddS24 B2D2(hetA2) 61.1 16.0 08/dd-2 D7 A1B2 84.2 37.3 08/dd-2 F1 B2 93.250.7 08/dd-2 G7 A1D2 92.6 49.7 08/dd-2 A1 B2D2 91.5 44.3 08/dd-2 F4 D284.4 45.7 08/dd-2 D5 wt 95.3 49.0

TABLE 18 SBEII allelic composition of mutants with multiple SBEIIa andSBEIIb null alleles Total number Number of wild-type Number of wild-typeof wild-type SBEIIa alleles present Total number of SBEIIb allelespresent Total number of SBEIIa and Plant on A, B and D genomes wild-typeSBEIIa on A, B and D genomes wild-type SBEIIb SBEIIb alleles AmyloseGenotype A A B B D D alleles present A A B B D D alleles present presentcontent % A1(+/−)B2D2 1 — — — — — 1/6 1 — 1 1 1 1 5/6 6/12 67% (pooled)A1B2D2(+/−) — — — — 1 — 1/6 — — 1 1 1 1 4/6 5/12 67% (pooled)A1B2(+/−)D2 — — 1 — — — 1/6 — — 1 1 1 1 4/6 5/12 67% (pooled) B2D2 1 1 —— — — 2/6 1 1 1 1 1 1 6/6 8/12 33.0-36.8 A1B2 — — — — 1 1 2/6 — — 1 1 11 4/6 6/12 33.9-34.9 A1D2 — — 1 1 — — 2/6 — — 1 1 1 1 4/6 6/12 32.2-37.0A1B1 — — — — 1 1 2/6 — — — — 1 1 2/6 4/12 34.1-34.7 A1D1 — — 1 1 — — 2/6— — 1 1 — — 2/6 4/12 32.8-38.7 A3D3 1 1 1 1 1 1 6/6 — — 1 1 — — 2/6 8/1230.8-31.6 A3B1 1 1 — — 1 1 4/6 — — — — 1 1 2/6 6/12 31.4 B1D3 1 1 — — 11 4/6 1 1 — — — — 2/6 6/12 30.3

TABLE 19 Further crosses between single and double null mutantsPotential Triple Parent Parent Mutant Observed progeny Cross 1 Code P1 2Code P2 genotype genotypes 08/hh-1  5-173 B1 08/i-G3 A1D2 A1B1D2 Allpossible single nulls and A1B2 double nulls identified, No triple nulls08/ii-1 20-365 B2 08/i-G3 A1D2 A1B2D2 All possible single nulls and B2D2double null identified, No triple nulls 08/ii-2 20-365 B2 08/i-C1 A1D2A1B2D2 All possible single nulls, B2D2 and A1D2 double nulls identified,no triple nulls 08/ii-3 20-365 B2 08/i-C8 A1D2 A1B2D2 All three doublenulls identified, but no triple nulls. 08/hh-4  5-173 B1 08/i-B12 A1D2A1B1D2 All three double nulls identified, but no triple nulls. 08/kk-2 5-563 A1 08/g-8G B2D2 A1B2D2 All possible single nulls and double nullsidentified, no triple nulls 08/kk-3  5-563 A1 08/g-A1 B2D2 A1B2D2 Allpossible single nulls and A1D2 and A1B2 double nulls identified, notriple nulls 08/kk-4  5-563 A1 08/g-D8 B2D2 A1B2D2 All single and doublenulls identified, No triple nulls 08/kk-6  5-563 A1 08/g-E10 B2D2 A1B2D2All possible single nulls and only B2D2 double nulls identified 08/ll-120-257 A1 08/g-E7 B2D2 A1B2D2 All possible single nulls and A1B2 andB2D2 identified, no triple nulls 08/ll-2 20-257 A1 08/g-8G B2D2 A1B2D2All possible single nulls and double nulls identified, no triple nulls08/ll-4 20-257 A1 08/g-D8 B2D2 A1B2D2 All possible single nulls and A1D2and B2D2 identified, no triple nulls 08/ll-6 20-257 A1 08/g-E10 B2D2A1B2D2 All possible single nulls and double nulls identified. No triplenulls 08/mm-1 19c-342  A2 08/d-C7 B2D2 A2B2D2 All possible single anddouble nulls identified. No triple nulls 08/mm-2 19-744 A2 08/f-C8 B2D2A2B2D2 No triple null 08/mm-3 19-744 A2 08/f-G9 B2D2 A2B2D2 No triplenull 08/mm-4 19-744 A2 08/e-F5 B2D2 A2B2D2 No triple null 08/mm-5 19-744A2 08/e-C11 B2D2 A2B2D2 No triple null 08/mm-6 19-744 A2 08/d-E8 B2D2A2B2D2 No triple null 08/mm-7 19-744 A2 08/d-D11 B2D2 A2B2D2 No triplenull 08/mm-8 19-342 A2 08/f-C8 B2D2 A2B2D2 No triple null 08/mm-9 19-342A2 08/f-C8 B2D2 A2B2D2 No triple null 08/mm-10 19-342 A2 08/f-C8 B2D2A2B2D2 No triple null 08/mm-11 19-342 A2 08/f-C8 B2D2 A2B2D2 No triplenull 08/mm-12 19-342 A2 08/f-C8 B2D2 A2B2D2 No triple null

TABLE 20 Observed frequency of genotypes of normally germinating grainfrom an A2B2D2 cross. Numbers in parentheses indicate the expectedfrequency based on Mendelian segregation WT A2 B2 D2 A2B2 A2D2 B2D2A2B2D2 08/mm1-4 69 7 9 4 2 3 2 0 08/mm1-6 56 16 10 9 2 1 2 0 08/mm1-7 4418 19 9 1 3 2 0 08mm1-5 54 13 15 10 1 1 2 0 08mm1-2 56 12 12 10 4 0 2 008/mm1-3 53 12 13 14 1 1 2 0 08/mm1-1 54 13 13 11 1 3 1 0 Total observed(expected) 386 (283) 90 (95) 91 (95) 67 (95) 12 (32) 12 (32) 13 (32) 0

TABLE 21 Further crosses between single and double null mutants Triplenull Screening Cross genotype status-(number designation Parent 1 Parent2 sought screened) 09/aa-1 08/k-F9 (18-111/3 x 5-173) 19-832 A3B1D1 285(only A3B1 (D1) A3B1 double null recovered) 09/bb-1 08/d-E8 (21-142 x12-801) 18-111/3 A3B2D2 285 (2 confirmed B2D2 (A3) A3B2D2 triple nulls).09/cc-1 08/b-12 (20-257 x 19-832) 19b-55 A1B3D1 285 (Only A1D1 (B3) B3D1double null recovered) 09/dd-1 08/i (20-257 x 12-801) 19b-55 A1B3D2 190(A1D2 and A1D2 (B3) A1B3 double nulls recovered) 09/ee-1 08/l-G9 (18-96x 5-173) 20-257 A1B1D3 190 (No B1D3 (A1) double nulls recovered) 09/ff-108/l-G9 (18-96 x 5-173) 19c-342 A2B1D3 190/190 B1D3 (A2) (Results notclear) 09/gg-1 08/j-D4 (18-111/3 x 18-96) 19b-55 A3B3D3 190 (1 A3D3 (B3)confirmed triple null)

TABLE 22 Putative double and triple null mutants in SBEIIa genesidentified in an initial screen using dominant markers Putative PutativeCross ID double triple Number Cross combination mutants mutants M76 TypeI-19.832 (D)/APACHE//Type 107 2 II-21.142(B)/Type I-20257(A) [08/h-92]M77 Type I-19.832 (D)/APACHE//Type 46 0 II-21.142(B)/Type I-20257(A)[08/h-111] M79 Type II-21.142(B)/Type I-20257 73 0 (A) [08/h-111]//TypeI-22.578 (D)/APACHE M81 Type II-21.142(B)/Type I-20257 14 0 (A)[08/h-111]//Type I-22.578 (D)/CHARA M74 Type I- 5.173 (B)//Type I-19.83216 0 (D)/Type II-20.257(A) [08/b12] M75 Type I-19.832 (D)/Type II-20.2571 0 (A) [08/b12]//Type I- 3.159 (B) M82 Type I-20.257 (A)/APACHE//Type128 4 II-12.801 (D)/Type II-21.142 (B) M83 Type I-20.257(A)/APACHE//Type 83 4 II-12.801 (D)/Type II-21.668 (B) M84 Type II-5.706 (D)/Type 36 1 II-21.668 (B)//Type I-20.257 (A)/APACHE M85 Type II-5.706 (D)/Type 171 8 II-21.668 (B)//Type I-20.257 (A)/CHARA M86 TypeII-12.801 (D)/Type 69 1 II-21.142 (B)//Type I-20.257 (A)/CHARA M78 TypeII-21.142(B)/Type I-20257 18 0 (A) [08/h-92]//Type I-19.832 (D)/CHARAM80 Type II-21.142(B)/Type I-20257 31 1 (A) [08/h-111]//Type I-19.832(D)/CHARA 793 21

TABLE 23 Starch characterisation of grain starch from transgenic wheatlines Amylose content Birefringence estimated Amylose content Enzyme nilpartial Full iodometrically determined by SEC Starch content Starchswelling Line ID targeted (%) (%) (%) (%) % (% w/w) power NB1Non-transformed 1.6 3.5 94.9 31.8 25.5 52.0 9.31 SBEIIa- SBEIIa 94.5 4.01.5 88.5 74.4 43.4 3.51 SBEIIb- SBEIIb 0.6 5.21 94.1 27.3 32.8 50.310.74 LSD (5%) 9.02 3.3 9.9 7.7 nd 4.9

TABLE 24 Molecular weight distribution of starch fractions from wheattransgenic lines Estimated Molecular Weight (kDa) Line Amylopectin HighMW amylose Low MW amylose Wild-type 45523.3 ± 2605.3 420.4 ± 23.2 8.56 ±0.2 (control) Reduced for 43646.4 ± 5259.6 409.6 ± 7.8  8.76 ± 0.1SBEIIb Reduced for 7166.1 ± 166.5 422.7 ± 26.8 9.70 ± 0.1 SBEIIa andSBEIIb

TABLE 25 RVA parameters of hp5′-SBEIIa transgenic wheat starch PastingFirst Final Peak Temp Line ID Construct Peak 1 Trough Break-downViscosity Setback Time (° C.) Control none 225.08 180.83 44.25 318137.17 10 85.3 SBEIIa hp5′-BEIIa 27.08 17.5 9.58 22.92 5.42 12.73 * *Starch from the reduced SBEIIa grain (line 85.2c) did not paste at thetemperature profile used in the RVA run.

TABLE 26 DSC parameters of gelatinisation peak of hp5′-SBEIIa transgenicwheat starch compared to the control NB1 Line ID Construct Onset ° C.Peak ° C. End ° C. Delta H NB1 Control 57.93 61.16 66.61 5.036 85.2chp5′-SBEIIa 57.38 63.51 72.61 2.385

TABLE 27 RS content in rolled and flaked grain products % RS Steamingtime (g/100 g Treatment No Line Roller width (Minute) product) HWFP03HAW Wide 60 13.3 HWFP05 HAW Wide 45 14.1 HWFP08 HAW Narrow 35 13.7HWFP09 HAW Wide 35 16.1 HWFP11 HAW Narrow 60 13.1 HWFP12 HAW Narrow 4511.4 HWFP01 Hartog Narrow 60 0.6 HWFP02 Hartog Wide 60 0.6 HWFP04 HartogWide 45 0.5 HWFP06 Hartog Narrow 45 0.4 HWFP07 Hartog Narrow 35 0.1HWFP10 Hartog Wide 35 0.2

TABLE 28 Resistant starch content in food products at varying level ofincorporation of high amylose wheat (HAW) Resistant Starch (g/100 gproduct) Incorporation level 0% 20% 40% 60% 80% 100% Type of productcontrol HAW Control HAW Control HAW Control HAW Control HAW Control HAWS & D bread NT NT 0.45 1.33 0.40 2.1 0.30 2.9 NT NT NT NT YAN 0.4 0 0.20.7 0 1.1 0.2 1.2 Spaghetti 0.3 1.3 0.1 2 0 2.9 0.1 4 0 6 Instant noodle0.4 0.4 0.3 0.8 0.2 1.4 0.2 1.6 NT NT NT NT Loaf bread NT NT 0.6 1.7 NTNT 0.6 3.7 NT NT 1 5.2 Flakes NT NT NT NT NT NT NT NT NT NT   0.2 16.1NT: Not tested

TABLE 29 Genome-specific primers SbeIIa SeqId Primer pair Covered exonsIIaA2_3 SbeIIa_A_deb2F/SbeIIa_A_deb5R 2, 3 IIaA6_7_8SbeIIa_A_deb4F/AR2aE8R07 6, 7, 8 IIaA12_14 Del5rev/Afor4 12, 14 IIaB2_3SbeIIa_Bdeb7F/BeIIaE3r 2, 3 IIaB12_14 BSNP17rev/Afor4 12, 14 IIaB21_22Sbe2a_Bfin-F2/BeIIaE22r 21, 22 IIaD2_3 SbeIIa_D_deb1F/SbeIIa_D_deb4R 2,3 IIaD6_7_8 SbeIIa_D_deb4F/AR2aE8R07 6, 7, 8 IIaD12_14 DSNP7rev/Afor412, 14 IIaD18_20 Sbe2a_Dfin-F1/Sbe2a_Dfin-R3 18, 20

BIBLIOGRAPHY

-   Abel et al., The Plant Journal 10: 981-991, 1996-   Altschul et al., Nucl. Acids Res. 25:3389, 1997-   Alvarez et al., Plant Cell 18: 1134-1151, 2006-   Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley    & Sons Inc, Chapter 15, 1994-1998-   Baga et al., Plant Mol Biol. 40(6): 1019-1030, 1999-   Batey and Curtin, Starch 48: 338-344, 1996-   Batey et al., Cereal Chemistry 74: 497-501, 1997-   Bechtold et al., C.R. Acad. Sci. Paris, 316:1194, 1993-   Bekes et al., Crit Care Med. 30(8): 1906-1907, 2002-   Blauth et al., Plant Physiology 125: 1396-1405, 2001-   Boyer and Preiss, Carbohydrate Research, 61: 321-334, 1978-   Boyer and Preiss, Plant Physiology 67: 1141-1145, 1981-   Boyer et al., Starch 32: 217-222, 1980-   Cao et al., Archives of Biochemistry and Biophysics, 373: 135-146,    2000-   Case et al., Journal of Cereal Science 27: 301-314, 1998-   Cheng et al., Plant Physiol 115: 971-980, 1997-   Comai et al., Plant J. 37: 778-786, 2004-   Craig et al., Plant Cell 10: 413-426, 1998-   Delwiche et al. Cereal Chem. 83(3):287-292, 2006-   Dowell et al Cereal Chem. 86(3):251-255, 2009-   Durai et al., Nucleic Acids Research 33(18): 5978-5990, 2005-   Eagles et al., Aust. J. Agric. Res. 52: 1 349-1356, 2001-   Fergason, Speciality Corns eds, CRC Press Inc. pp 55-77, 1994-   Fisher et al., Plant Physiol 102: 1045-1046, 1993-   Fisher et al., Journal Plant Physiol. 108(3): 1313-1314, 1995-   Fromm et al., Proc. Natl. Acad. Sci., U.S.A, 82: 5824, 1985-   Fuwa et al., Starch-Stärke 51: 147-151, 1999-   Gao et al., Plant Cell, 10: 399-412, 1998-   Gao et al., Plant Physiol 114: 69-78, 1997-   Goddard et al., Am J Clin Nutr 39: 388-392, 1984-   Gras et al., In: Proc. ICC 11th Int. Cereal and Bread Congress.    RACI: Melbourne, 2001b-   Green et al., Plant Physiol 114: 203-212, 1997-   Harayama, Trends Biotechnol. 16: 76-82, 1998-   Hartmann and Endres, Manual of Antisense Methodology, Kluwer, 1999-   Haseloff and Gerlach, Nature 334: 585-591, 1988-   Hayashi et al., Effects of ion beam irradiation on mutation    induction in rice. Cyclotrons and Their Applications 2007,    Eighteenth International Conference 237-239, 2007-   Hedman and Boyer, Biochemical Genetics 20: 483-492, 1982-   Henikoff et al., Plant Physiol. 135: 630-636, 2004-   Hinchee et al., Biotech., 6: 915, 1988-   James et al., Plant Cell, 7: 417-429, 1995-   Jobling et al., Plant Journal 18: 163-171, 1999-   Kanna and Daggard, Plant Cell Rep 21: 429-436, 2003-   Kawasaki et al., Mol Gen Genet. 237(1-2): 10-6, 1993-   Kazama et al., Plant Biotechnology 25: 113-117, 2008-   Kim et al., Plant Physiol. 121(1): 225-236, 1999-   Kim et al., Gene. 216(2): 233-43, 1998a-   Klein et al., Nature 327: 70, 1987-   Klein et al., Nature 327: 70, 1987-   Konik-Rose et al., Starch-Stärke, 53: 14-20, 2001-   Krueger et al., Cereal Chemistry 64: 187-190, 1987-   Kubo et al., Plant Physiology, 121: 399-409, 1999-   Langridge et al., Aust. J. Agric. Res. 52: 1043-1077, 2001-   Lazo et al., Biotechnology (N Y). 9(10): 963-967, 1991-   Le Provost et al., Trends in Biotechnology 28(3): 134-141, 2009-   Lee et al., Cell 75: 843-854, 1993-   Li et al., Plant Physiology, 120: 1147-1155, 1999a-   Li et al., Theoretical and Applied Genetics, 98: 1208-1216, 1999b-   Li et al., Plant Physiology, 123: 613-624, 2000-   Liu et al., Biotechnology and Bioengineering, 106: 97-105, 2010-   Mann et al., In ‘Workshop on gluten proteins’ Eds. Lafiandra et al.,    The Royal Society of Chemistry: Cambridge, UK, pp. 215-218, 2003-   Mann, New crop phenomenon in wheat and the mechanisms involved, PhD    thesis, University of Reading, UK, 2002-   McCallum et al., Nat Biotechnol 18:455-457, 2000-   McClure, J. Near Infrared Spectrosc. 11, 487-518, 2003-   McCreery and Helentjaris, Methods in Molecular Biology, Vol. 28:    Protocols for nucleic acid analysis by non-radioactive probes,    Humana Press Inc., Totawa, N.J., pp 67-71, 1994-   Mizuno et al., Journal of Biological Chemistry 268: 19084-19091,    1993-   Morell et al., Electrophoresis 19: 2603-2611, 1998-   Morell et al., Plant J. 34: 173-185, 2003-   Morell et al., Plant Physiol 113: 201-208, 1997-   Morrison and Laignelet, J Cereal Sci, 1: 9-20, 1983-   Moullet et al., Theor Appl Genet 99: 305-313, 1999-   Myers et al., Plant Physiology, 122: 989-997, 2000-   Nair et al., Plant Sci 122: 153-163, 1997-   Nakamura, Plant Cell Physiology 43: 718-725, 2002-   Needleman and Wunsch, J. Mol. Biol. 48: 443-453, 1970-   Niedz et al., Plant Cell Reports, 14: 403, 1995-   Nishi et al., Plant Physiology 127: 459-472, 2001-   Ow et al., Science, 234: 856, 1986-   Palatnik et al., Nature 425: 257-263, 2003-   Parizotto et al., Genes Dev 18: 2237-2242, 2004-   Perriman et al., Gene, 113: 157-163, 1992-   Potrykus et al., Mol. Gen. Genet., 199: 183, 1985-   Prasher et al., Biochem. Biophys. Res. Comm. 126: 1259-68, 1985-   Rahman et al., Genome 40: 465-474, 1997-   Rahman et al., Theor. Appl. Genet. 98: 156-163, 1999-   Rahman et al., Aust. J. Plant Physiol. 22: 793-803, 1995-   Rahman et al., Plant Physiol 125: 1314-1324, 2001-   Regina et al., J Exp Bot. 61(5): 1469-1482, 2010-   Regina et al., Funct Plant Biol, 31: 591-601, 2004-   Regina et al., Planta 222: 899-909, 2005-   Regina et al., Proc Natl Acad Sci USA, 103: 3546-3551, 2006-   Repellin et al., Plant Gene Reg pp. 97-094, 1997-   Safford et al., Carbohydrate Polymers 35: 155-168, 1998-   Schulman and Kammiovirta, Starch 43: 387-389, 1991-   Schwab et al., Plant Cell 18: 1121-1133, 2006-   Schwall et al., Nature Biotechnology 18: 551-554, 2000-   Sestili et al., Mol Breeding 25:145-154, 2010-   Sharp et al., Aust J Agric Res 52: 1357-1366, 2001-   Shimamoto et al., Nature 338: 274-276, 1989-   Shippy et al., Mol. Biotech. 12: 117-129, 1999-   Shure et al., Cell 35: 225-233, 1983-   Sidebottom et al., Journal of Cereal Science 27: 279-287, 1998-   Sissons et al., Journal of the Science of Food and Agriculture 87:    1874-1885, 2007-   Slade and Knauf, Transgenic Res. 14: 109-115, 2005-   Smewing, The measurement of dough and gluten extensibility using the    SMS/Kieffer rig and the TA.TX2 texture analyzer handbook, SMS Ltd:    Surrey, UK, 1995-   Smith et al., Nature, 407: 319-320, 2000-   Sparks and Jones in Transgenic Crops of the World—Essential    protocols, Ed. IP Curtis, Kluwer Academic Publishers, Dordrecht,    Netherlands, pp 19-34, 2004-   Stacey and Isaac, Methods in Molecular Biology, Vol. 28: Protocols    for nucleic acid analysis by non-radioactive probes, Humana Press    Inc., Totawa, N.J., pp 9-15, 1994-   Stalker et al., Science, 242: 419, 1988-   Sun et al., Plant Physiol 118: 37-49, 1998-   Takeda et al., Carbohydrate Research 240: 253-262, 1993a-   Takeda et al., Carbohydrate Research 246: 273-281, 1993b-   Tanaka et al., Nucl. Acids Res. 18: 6767-6770, 1990-   Tetlow et al., Plant Cell 16: 694-708, 2004-   Tetlow et al., Plant Physiol 146: 1878-1891, 2008-   Thillet et al., J. Biol. Chem., 263: 12500, 1988-   Vasil, Bio/Technol. 8: 429-434, 1990-   Vogel et al., Fermentation and Biochemical Engineering Handbook:    Principles, Process Design, and Equipment, Noyes Publications, Park    Ridge, N.J., USA, 1996-   Waterhouse et al., Proc. Natl. Acad. Sci. U.S.A. 95: 13959-13964,    1998-   Weir et al., Aust J Plant Physiol 28: 807-818, 2001-   Wu et al., Plant Cell Rep 21: 659-668, 2003-   Yamamori et al., Theor Appl Genet, 101: 21-29, 2000-   Nakamura and Yamanouchi, Plant Physiol. 99(3): 1265-1266, 1992-   Yu et al., Science 307: 932-935, 2005-   Zwar and Chandler, Planta 197: 39-48, 1995

The claims defining the invention are as follows:
 1. Wheat grain ofTriticum aestivum, the grain comprising an embryo, an endosperm andstarch, wherein (a) the embryo is homozygous for a loss of functionmutation in each of three starch branching enzyme IIa (SBEIIa) genes,wherein said SBEIIa genes are an SBEIIa-A gene, an SBEIIa-B gene and anSBEIIa-D gene, and wherein the loss of function mutation in at least oneof the SBEIIa genes is a point mutation or a deletion of 1-30base-pairs, (b) the grain has a germination rate of between 70% and 100%relative to the germination rate of wild-type wheat grain, and (c) thestarch comprises amylose such that the grain has an increased amylosecontent as a proportion of the starch in the grain relative to wild-typewheat grain.
 2. The wheat grain of claim 1, wherein the starch comprisesamylose such that the grain has an amylose content of at least 50% (w/w)as a proportion of the starch in the grain, as measured by an iodinebinding assay.
 3. The wheat grain of claim 1, wherein the starchcomprises amylose such that the grain has an amylose content of at least60% (w/w) as a proportion of the starch in the grain, as measured by aniodine binding assay.
 4. The wheat grain of claim 1, wherein the starchcomprises amylose such that the grain has an amylose content of at least67% (w/w) as a proportion of the starch in the grain, as measured by aniodine binding assay.
 5. The wheat grain of claim 1, wherein the loss offunction mutation in at least one of the SBEIIa genes is a pointmutation.
 6. The wheat grain of claim 1, wherein the point mutation isin the SBEIIa-A gene.
 7. The wheat grain of claim 6, wherein theSBEIIa-B gene and the SBEIIa-D gene each comprise a deletion mutationwhich deletes at least part of the gene, or deletes the whole of thegene.
 8. The wheat grain of claim 1, wherein the point mutation is inthe SBEIIa-B gene.
 9. The wheat grain of claim 8, wherein the SBEIIa-Agene and the SBEIIa-D gene each comprise a deletion mutation whichdeletes at least part of the gene, or deletes the whole of the gene. 10.The wheat grain of claim 1, wherein the point mutation is in theSBEIIa-D gene.
 11. The wheat grain of claim 10, wherein the SBEIIa-Agene and the SBEIIa-B gene each comprise a deletion mutation whichdeletes at least part of the gene, or deletes the whole of the gene. 12.The wheat grain of claim 1, wherein the loss of function mutation thatis a point mutation or a deletion of 1-30 base-pairs is a splice-sitemutation or a premature translation termination mutation.
 13. The wheatgrain of claim 1, wherein the starch of the wheat grain has one or moreof the properties selected from the group consisting of: (i) comprisingat least 2% resistant starch, (ii) a low relative glycemic index, (iii)a reduced level of amylopectin, (iv) distorted starch granules, (v)reduced starch granule birefringence, (vi) a reduced swelling volume,(vii) a modified chain length distribution and/or branching frequency,(viii) an increased end of gelatinization temperature and increased peakviscosity, (ix) increased molecular weight of amylopectin, (x) amodified percentage of crystalline starch, and (xi) a modifiedpercentage of A-type or B-type crystalline starch, wherein each of theproperties are relative to starch from grain of the wheat cultivarSunstate.
 14. The wheat grain of claim 1, wherein the starch of thegrain comprises 3% to 18%, or 5% to 15%, resistant starch, by weight.15. A wheat plant which produces grain according to claim 1, wherein thewheat plant comprises the loss of function mutations in each of thethree SBEIIa genes.
 16. The wheat plant of claim 15, wherein said wheatplant is a progeny of a cross of: i) a wheat plant comprising a loss offunction mutation in either an SBEIIa-A gene or an SBEIIa-B gene or anSBEIIa-D gene, wherein the loss of function mutation is a point mutationor a deletion of 1-30 base-pairs, and ii) a wheat plant comprising lossof function mutations in either the SBEIIa-A and SBEIIa-B genes, or theSBEIIa-A and SBEIIa-D genes, or the SBEIIa-B and SBEIIa-D genes, saidloss of function mutations being in genes on different genomes from thegene comprising said loss of function mutation that is a point mutationor a deletion of 1-30 base-pairs.
 17. The wheat plant of claim 15,wherein the loss of function mutation that is a point mutation or adeletion of 1-30 base-pairs is a splice-site mutation or a prematuretranslation termination mutation.
 18. The wheat plant of claim 15,wherein the loss of function mutation that is a point mutation or adeletion of 1-30 base-pairs is a frameshift mutation.
 19. The wheatplant of claim 15, wherein the loss of function mutation that is a pointmutation or a deletion of 1-30 base-pairs was produced by ethylmethanesulfonate (EMS) mutagenesis.
 20. The wheat plant of claim 15,wherein the loss of function mutation that is a point mutation or adeletion of 1-30 base-pairs was produced by sodium azide mutagenesis.21. A process for producing a milled product, comprising the steps of:(i) obtaining wheat grain of claim 1; and (ii) milling the grain,thereby producing the milled product.
 22. A milled product produced fromthe grain of claim 1, the milled product comprising DNA which comprisesthe loss of function mutations in each of the three SBEIIa genes, andstarch, wherein the starch comprises an increased amylose content as aproportion of the starch relative to wild-type wheat starch.
 23. Themilled product of claim 22, wherein the starch has one or more of theproperties selected from the group consisting of: (i) comprising atleast 2% resistant starch, (ii) a low relative glycemic index, (iii) areduced level of amylopectin, (iv) distorted starch granules, (v)reduced starch granule birefringence, (vi) a reduced swelling volume,(vii) a modified chain length distribution and/or branching frequency,(viii) an increased end of gelatinization temperature and increased peakviscosity, (ix) increased molecular weight of amylopectin, (x) amodified percentage of crystalline starch, and (xi) a modifiedpercentage of A-type or B-type crystalline starch, wherein each of theproperties are relative to starch from grain of the wheat cultivarSunstate.
 24. The milled product of claim 22, wherein the starch of themilled product comprises 3% to 18%, or 5% to 15%, resistant starch, byweight.
 25. A food product comprising the milled product of claim 22.